Anti-IL-9 antibody formulations

ABSTRACT

The present invention provides liquid formulations of antibodies or antibody fragments that immunospecifically bind to an IL-9 polypeptide, which formulations exhibit stability, low to undetectable levels of aggregation, and very little to no loss of the biological activities of the antibodies or antibody fragments, even during long periods of storage. In particular, the present invention provides liquid formulations of antibodies or fragments thereof that immunospecifically bind to an IL-9 polypeptide, which formulations are substantially free of surfactants, sugars, sugar alcohols, amino acids other than histidine (preferably with pKa values of less than 5 and above 7), and/or other common excipients. Furthermore, the invention provides methods of preventing, treating or ameliorating a disease or disorder associated with or characterized by aberrant expression and/or activity of an IL-9 polypeptide, a disease or disorder associated with or characterized by aberrant expression and/or activity of the IL-9R or one or more subunits thereof, an autoimmune disease, an inflammatory disease, a proliferative disease, or an infection (preferably, a respiratory infection), or one or more symptoms thereof, utilizing the liquid formulations of the present invention.

This application claims the benefit of U.S. Provisional Application No.60/561,845, filed Apr. 12, 2004, which is incorporated by referenceherein in its entirety.

1. INTRODUCTION

The present invention relates to high concentration liquid formulationsof antibodies or fragments thereof that immunospecifically bind to anIL-9 polypeptide, which formulations exhibit stability, low toundetectable levels of antibody fragmentation, low to undetectablelevels of aggregation, and very little to no loss of the biologicalactivities of the antibodies, even during long periods of storage. Inparticular, the present invention relates to liquid formulations ofantibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide, which formulations aresubstantially free of surfactants, sugars, sugar alcohols and/or aminoacids other than histidine. The present invention also relates tomethods of preventing, treating, managing or ameliorating symptomsassociated with an inflammatory disorder (e.g., asthma), or arespiratory infection, utilizing high concentration liquid formulationsof antibodies or (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide.

2. BACKGROUND OF THE INVENTION

Currently, many antibodies are provided as lyophilized formulations.Lyophilized formulations of antibodies have a number of limitations,including a prolonged process for lyophilization and resulting high costfor manufacturing. In addition, a lyophilized formulation has to bereconstituted aseptically and accurately by healthcare practitionersprior to administering to patients. The reconstitution step itselfrequires certain specific procedures: (1) a sterile diluent (i.e., waterfor intravenous administration and 5% dextrose in water forintramuscular administration) is added to the vial containinglyophilized antibody, slowly and aseptically, and the vial must beswirled very gently for 30 seconds to avoid foaming; (2) thereconstituted antibody may need to stand at room temperature for aminimum of 20 minutes until the solution clarifies; and (3) thereconstituted preparation must be administered within six (6) hoursafter the reconstitution. Such reconstitution procedure is cumbersomeand the time limitation after the reconstitution can cause a greatinconvenience in administering the formulation to patients, leading tosignificant waste, if not reconstituted properly or if the reconstituteddose is not used within six (6) hours and must be discarded.

Thus, a need exists for liquid formulations of antibodies, inparticular, anti-IL-9 antibodies, at a concentration comparable to orhigher than the reconstituted lyophilized formulations so that there isno need to reconstitute the formulation prior to administration. Thisallows healthcare practitioners much quicker and easier administrationof antibodies to a patient.

Prior liquid antibody preparations have short shelf lives and may losebiological activity of the antibodies resulting from chemical andphysical instabilities during the storage. Chemical instability may becaused by deamidation, racemization, hydrolysis, oxidation, betaelimination or disulfide exchange, and physical instability may becaused by antibody denaturation, aggregation, precipitation oradsorption. Among those, aggregation, deamidation and oxidation areknown to be the most common causes of the antibody degradation (Wang etal., 1988, J. of Parenteral Science & Technology 42(Suppl):S4-S26;Cleland et al., 1993, Critical Reviews in Therapeutic Drug CarrierSystems 10(4):307-377). Thus, there is a need for a stable liquidformulation of antibodies, in particular, stable liquid anti-IL-9antibodies.

3. SUMMARY OF INVENTION

The present invention is based, in part, on the development of highconcentration liquid formulations of antibodies (including antibodyfragments thereof) that immunospecifically bind to an IL-9 polypeptide,which formulations exhibit, in the absence of surfactants, sugars, sugaralcohols, and amino acids other than histidine (preferably, free ofamino acids with pKa values of less than 5 and above 7), stability, lowto undetectable levels of antibody fragmentation and/or aggregation, andvery little to no loss of the biological activities of the antibodies(including antibody fragments thereof) during manufacture, preparation,transportation, and storage. The liquid formulations of the presentinvention facilitate the administration of antibodies (includingantibody fragments thereof) that immunospecifically bind to an IL-9polypeptide for the prevention, treatment, management and ameliorationof diseases or disorders associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, diseases or disordersassociated with or characterized by aberrant expression and/or activityof the IL-9 receptor (“IL-9R”) or one or more subunits thereof,autoimmune diseases, inflammatory diseases, proliferative diseases, orinfections (preferably, respiratory infections), or one or more symptomsthereof (e.g., wheezing). Examples of autoimmune diseases include, butare not limited to: diabetes, Hashimoto's disease, autoimmune adrenalinsufficiency, pure red cell anemia, multiple sclerosis, rheumatoidcarditis, systemic lupus erythematosus, rheumatoid arthritis, chronicinflammation, Sjogren's syndrome polymyositis, dermatomyositis andscleroderma. Examples of inflammatory disorders include, but are notlimited to, asthma and allergic reactions (Types I-IV). Examples ofrespiratory infections include, but are not limited to, infections ofthe upper and lower respiratory tracts, including viral infections,bacterial infections and/or fungal infections. Examples of viralinfections include parainfluenza virus infection, influenza virusinfection, metapenumovirus infection, or respiratory syncytial virus(RSV) infection. The antibody formulations of the invention may also beused to treat subjects that have or previously had bronchopulmonarydysplasia, congenital heart disease, cysteic fibrosis or acquired orcongenital immunodeficiency. In particular, the liquid formulations ofthe present invention enable a healthcare professional to quicklyadminister a sterile dosage of an antibody (including antibody fragmentthereof) that immunospecifically binds to an IL-9 polypeptide withouthaving to accurately and sterilely reconstitute the antibody (includingantibody fragment thereof) prior to administration.

The present invention provides liquid formulations substantially free ofsurfactants, said formulations preferably comprising histidine at aconcentration in the range from about 5 to about 25 mM or higher(preferably at a concentration of 10 mM), NaCl at a concentration in therange from about 100 to about 200 mM (preferably at a concentration of150 mM) and antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide at a concentration of 50mg/ml or higher (although, in certain embodiments, the concentration ofthe antibody is lower than 50 mg/ml). The present invention alsoprovides liquid formulations substantially free of surfactants, sugars,sugar alcohols, and/or amino acids (preferably, free of amino acids witha pKa value of less than 5 and above 7, particularly amino acids otherthan histidine), said formulations having a pH ranging from about 5.0 toabout 7.0, preferably about pH 6.0, and comprising histidine, and aconcentration of 50 mg/ml or higher of antibodies (including antibodyfragments thereof) that immunospecifically bind to an IL-9 polypeptide.The liquid formulations of the present invention may further compriseone or more excipients such as a saccharide, an amino acid (e.g.,arginine, lysine, and methionine) and a polyol. In a preferredembodiment, a liquid formulation of the present invention compriseshistidine and a concentration of 95 mg/ml or higher of antibodies(including antibody fragments thereof) that immunospecifically bind toan IL-9 polypeptide, and said formulation is substantially free ofsurfactants, sugars, sugar alcohols, and/or amino acids other thanhistidine.

The present invention encompasses stable liquid formulations of 4D4,4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4 (for amino acid sequences, see U.S. patentapplication Ser. No. 60/477,797, filed Jun. 10, 2003, and U.S. patentapplication Ser. No. 10/823,253 to be filed concurrently herewith onApr. 12, 2004, entitled “Recombinant IL-9 Antibodies and Uses Thereof,”both of which are incorporated by reference herein in their entireties)which exhibit low to undetectable levels of antibody aggregation and/orfragmentation with very little to no loss of the biological activitiesof 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 during manufacture, preparation,transportation, and long periods of storage. The present invention alsoencompasses stable liquid formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide and have increased invivo half-lives relative to known antibodies such as, e.g., 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4, said formulations exhibiting low toundetectable levels of antibody aggregation and/or fragmentation, andvery little to no loss of the biological activities of the antibodies(including antibody fragments thereof). The present invention alsoencompasses stable liquid formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodies(including antibody fragments thereof) comprising a variable heavy (VH)and/or variable light (VL) domain having the amino acid sequence of theVH and/or VL domain of 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4, saidformulations exhibiting low to undetectable levels of antibodyaggregation and/or fragmentation, and very little to no loss of thebiological activities of the antibodies (including antibody fragmentsthereof). The present invention further encompasses stable liquidformulations of antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide, said antibodies(including antibody fragments thereof) comprising one or more VHcomplementarity determining regions (CDRs) and/or one or more VL CDRshaving the amino acid sequence of one or more VH CDRs and/or VL CDRslisted in Table 1, infra, said formulations exhibiting low toundetectable levels of antibody aggregation and/or fragmentation, andvery little to no loss of the biological activities of the antibodies(including antibody fragments thereof).

TABLE 1 Residues that are different between each amino acid sequenceencoding the various CDRs appear in bold, underlined font. Antibody NameVH Domain VH CDR1 VH CDR2 VH CDR3 VL Domain VL CDR1 VL CDR2 VL CDR3 4D4SEQ. ID NO.: GYTF TG YWI E I LPGSGTTN ADYYGSD Y V SEQ. ID NO.: KASQHVGTH S TSYRYS Q H FYSYPLT 7 E YNEKFKG KEDY 8 VT (SEQ. ID (SEQ. ID(SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID NO.: 5) NO.: 6) NO. 1): NO.: 2) NO.:3) NO.: 4) 4D4 H2-1 SEQ. ID NO.: GYTF TG YWI E W LPGSGTT ADYYGSDYV SEQ.ID NO.: K ASQHVGTH S TSYRYS Q H FYSYPLT D11 9 E NYNEKFKG KFDY 8 VT (SEQ.ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID NO.: 5) NO.: 6) NO.: 1)NO.: 10) NO.: 3) NO.: 4) 4D4com-XF-9 SEQ. ID NO.: GYTF TY YWI E WLPGSGTT ADYYGSD H V SEQ. ID NO.: L ASQHVGTH G TSYRYS Q H FYDYPLT 15 ENYNEKFKG KFDY 16 VT (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ.ID NO.: 13) NO.: 14) NO.: 6) NO.: 11) NO.: 10) NO.: 12) 4D4com-2F9 SEQ.ID NO.: GYTF TG YWI E W LPGSGTT ADYYGSD H V SEQ. ID NO.: K ASQHVGTH GTSYRYS Q H FYEYPLT 17 E NYNEKFKG KFDY 18 VT (SEQ. ID (SEQ. ID (SEQ. ID(SEQ. ID (SEQ. ID (SEQ. ID NO.: 14) NO.: 6) NO.: 1) NO.: 10) NO.: 12)NO.: 4) 7F3 SEQ. ID NO.: GGTF SG YWI E I LPGSGTTN ADYYGSD Y V SEQ. IDNO.: K ASQHVGTH S TSYRYS Q Q FYEYPLT 21 E YNEKFKG KFDY 22 VT (SEQ. ID(SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID NO.: 5) NO.: 20) NO.: 19)NO.: 2) NO.: 3) NO.: 4) 71A10 SEQ. ID NO.: GGTF SG YWI E I LPGSGTTNADYYGSD Y V SEQ. ID NO.: K ASQHVGTH S TSYRYS Q Q FYEYPLT 23 E PNEKFKGKEDY 24 VT (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID NO.: 5)NO.: 20) NO.: 19) NO.: 2) NO.: 3) NO.: 4) 7F3 22D3 SEQ. ID NO.: GGTF SGYWI E I LPGSGTTN ADYYGSD Y V SEQ. ID NO.: K ASQHVGTH G TSYRYS Q QFYEYPLT 21 E YNEKFKG KFDY 25 VT (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID(SEQ. ID (SEQ. ID NO.: 14) NO.: 20) NO.: 19) NO.: 2) NO.: 3) NO.: 4)7F3com-2H2 SEQ. ID NO.: GGTF SY YWI E I LPGSGTTN ADYYGSD Y V SEQ. IDNO.: K ASQHVITH G TSYSYS Q Q FYEYPLT 27 E PNEKFKG KFDY (SEQ. 28 VT (SEQ.ID (SEQ. ID (SEQ. ID (SEQ. ID ID NO.: 3) (SEQ. ID NO.: 14) NO.: 20) NO.:26) NO.: 2) NO.: 4) 7F3com-3H5 SEQ. ID NO.: GGTF SG YWI E I LPGSGTTNADYYGSD Y V SEQ. ID NO.: K ASQHVGTH G TSYRYS Q Q FYEYPLT 29 E PNEKFKGKFDY 30 VT (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID NO.:14) NO.: 20) NO.: 19) NO.: 2) NO.: 3) NO.: 4) 7F3com-3D4 SEQ. ID NO.:GGTF SY YWI E I LPGSGTTN ADYYGSD Y V SEQ. ID NO.: K ASQHVITH G TSYRYS QQ FYEYPLT 31 E PNEKFKG KFDY 32 VT (SEQ. ID (SEQ. ID (SEQ. ID (SEQ. ID(SEQ. ID (SEQ. ID NO.: 14) NO.: 20) NO.: 26) NO.: 2) NO.: 3) NO.: 4)

The present invention encompasses liquid formulations of antibodies(including antibody fragments thereof) that immunospecifically bind toan IL-9 polypeptide, said formulations having stability at 38-42° C. asassessed by high performance size exclusion chromatography (HPSEC). Theliquid formulations of the present invention exhibit stability, asassessed by HPSEC, at temperature ranges of 38-42° C. for at least 15days but no more than 25 days; at temperature ranges of 20-24° C. for atleast 6 months but not more than 1.5 years; and at temperature ranges of2-8° C. (especially at 4° C.) for at least 1.5 years, at least 2 years,at least 2.5 years, or at least 3 years. The present invention alsoencompasses liquid formulations of antibodies (including antibodyfragments thereof) that immunospecifically bind to an IL-9 polypeptide,said formulations having low to undetectable levels of antibodyaggregation as measured by HPSEC. In a preferred embodiment, the liquidformulations of the present invention exhibit stability at 38-42° C. forat least 15 days and exhibit low to undetectable levels of antibodyaggregation as measured by HPSEC and, further, exhibit very little to noloss of the biological activity of the antibodies (including antibodyfragments thereof) of the formulation compared to the referenceantibodies as measured by antibody binding assays such as, e.g., ELISAs.

The present invention provides methods for preparing liquid formulationsof an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide, said methods comprisingconcentrating a fraction containing the purified antibody to a finalantibody concentration ranging from about 1 mg/ml, about 5 mg/ml, about10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml,about 90 mg/ml, about 100 mg/ml, about 150 mg/ml, about 175 mg/ml, orabout 200 mg/ml using a semipermeable membrane with an appropriatemolecular weight (MW) cutoff (e.g., a 30 kD cutoff for whole antibodymolecules and F(ab′)₂ fragments; and a 10 kD cutoff for antibodyfragments such as Fab fragments) and diafiltering the concentratedantibody fraction into the formulation buffer using the same membrane.The formulation buffer of the present invention comprises histidine at aconcentration ranging from about 1 mM to about 100 mM, preferably fromabout 5 mM to about 50 mM, more preferably about 10 mM to about 25 mM.The formulation buffer of the present invention further comprises NaClat a concentration ranging from about 10 mM to about 200 mM, from about50 to about 200 mM, from about 100 to about 150 mM, more preferablyabout 150 mM. The pH of the formulation may range from about 5.0 toabout 7.0, preferably 5.5 to about 6.5, more preferably about 5.8 toabout 6.2, and most preferably about 6.0. To obtain an appropriate pHfor a particular antibody, it is preferable that histidine (and glycine,if added) is first dissolved in water to obtain a buffer solution withhigher pH than the desired pH and then the pH is brought down to thedesired level by the addition of HCl. This way, the formation ofinorganic salts (e.g., the formation of NaCl when, e.g., histidinehydrochloride is used as the source of histidine and the pH is raised tothe desired level by the addition of NaOH) can be avoided.

The liquid formulations of the present invention may be sterilized bysterile filtration using a 0.2μ filter. Sterilized liquid formulationsof the present invention may be administered to a subject for theprevention, treatment, management and amelioration of a disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof. The liquidformulations of the present invention may be administered in combinationwith other therapies (e.g., prophylactic or therapeutic agents otherthan antibodies that immunospecifically bind to an IL-9 polypeptide,such as anti-inflammatory agents, immunomodulatory agents andanti-cancer agents).

The present invention also provides kits comprising the liquidformulations of antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide for use by, e.g., ahealthcare professional. The present invention further provides methodsof preventing, treating, managing or ameliorating a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms of any of the foregoing.The liquid formulations of the invention can be administeredparenterally (e.g., intradermally, intramuscularly, intraperitoneally,intravenously and subcutaneously) orally, or intranasally to a subjectto prevent, treat, manage or ameliorate a disease or disorder associatedwith or characterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms of any of the foregoing. The liquid formulations of thepresent invention can also be used to diagnose, detect or monitordisease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, diseases or disordersassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, autoimmune diseases,inflammatory diseases, proliferative diseases, or infections(preferably, respiratory infections), or one or more symptoms thereof.

3.1. Terminology

All liquid formulations of antibodies and/or antibody fragments thatimmunospecifically bind to an IL-9 polypeptide described above areherein collectively referred to as “liquid formulations of theinvention,” “antibody liquid formulations of the invention,” “antibodyformulations of the invention,” “liquid formulations of antibodies orfragments thereof that immunospecifically bind to an IL-9 polypeptide,”or “liquid formulations of anti-IL-9 antibodies.”

As used herein the term “aberrant” refers to a deviation from the norm,e.g., the average healthy subject and/or a population of average healthysubjects. The term “aberrant expression,” as used herein, refers toabnormal expression of a gene product (e.g., RNA, protein, polypeptide,or peptide) by a cell or subject relative to a normal, healthy cell orsubject and/or a population of normal, healthy cells or subjects. Suchaberrant expression may be the result of the amplification of the gene.In a specific embodiment, the term “aberrant expression” refers toabnormal expression of IL-9 and/or an IL-9R or subunit thereof by a cellor subject relative to the expression of the gene product by a normal,healthy cell or subject and/or a population of normal, healthy cells orsubjects and encompasses the expression of an IL-9 and/or an IL-9R orsubunit thereof gene product at an unusual location within the cell orsubject, the expression of an IL-9 and/or an IL-9R or subunit thereofgene product at an altered level in the cell or subject, the expressionof a mutated IL-9 and/or an IL-9R or subunit thereof gene product, or acombination thereof. The term “aberrant activity,” as used herein,refers to an altered level of a gene product, the increase of anactivity by a gene product, or the loss of an activity of a gene productin a cell or subject relative to a normal, healthy cell or subjectand/or a population of normal healthy cells or subjects. In specificembodiments, the term “aberrant activity” refers to an IL-9 and/or IL-9Ror subunit thereof activity that deviates from that normally found in ahealthy cell or subject and/or a population of normal, healthy cells orsubjects (e.g., an increase in IL-9's affinity for the IL-9R). Examplesof IL-9 activities include, but are not limited to, the phosphorylationof the IL-9R, the activation of Jak3, the activation of MEK, theactivation of STAT-1, and the activation of STAT-3.

As used herein, the term “about” in the context of a given numeratevalue or range refers to a value or range that is within 20%, preferablywithin 10%, and more preferably within 5% of the given value or range.

As used herein, the term “analog” in the context of a proteinaceousagent (e.g., proteins, polypeptides, peptides, and antibodies) refers toa proteinaceous agent that possesses a similar or identical functions asa second proteinaceous agent but does not necessarily comprise a similaror identical amino acid sequence of the second proteinaceous agent, orpossess a similar or identical structure of the second proteinaceousagent. A proteinaceous agent that has a similar amino acid sequencerefers to a second proteinaceous agent that satisfies at least one ofthe following: (a) a proteinaceous agent having an amino acid sequencethat is at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99% identical to the amino acid sequence of a second proteinaceousagent; (b) a proteinaceous agent encoded by a nucleotide sequence thathybridizes under stringent conditions to a nucleotide sequence encodinga second proteinaceous agent of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least 100 contiguous aminoacid residues, at least 125 contiguous amino acid residues, or at least150 contiguous amino acid residues; and (c) a proteinaceous agentencoded by a nucleotide sequence that is at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% identical to the nucleotidesequence encoding a second proteinaceous agent. A proteinaceous agentwith similar structure to a second proteinaceous agent refers to aproteinaceous agent that has a similar secondary, tertiary or quaternarystructure to the second proteinaceous agent. The structure of aproteinaceous agent can be determined by methods known to those skilledin the art, including but not limited to, peptide sequencing, X-raycrystallography, nuclear magnetic resonance, circular dichroism, andcrystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul,1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score-50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,the NCBI website). Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the term “analog” in the context of a non-proteinaceousanalog refers to a second organic or inorganic molecule which possessesa similar or identical function as a first organic or inorganic moleculeand is structurally similar to the first organic or inorganic molecule.

As used herein, the terms “antagonist” and “antagonists” refer to anyprotein, polypeptide, peptide, peptidomimetic, glycoprotein, antibody,antibody fragment, carbohydrate, nucleic acid, organic molecule,inorganic molecule, large molecule, or small molecule that blocks,inhibits, reduces or neutralizes the function, activity and/orexpression of another molecule. In various embodiments, an antagonistreduces the function, activity and/or expression of another molecule byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99% relative to acontrol such as phosphate buffered saline (PBS).

The term “antibody fragment” as used herein refers to a fragment of anantibody that immunospecifically binds to an IL-9 polypeptide. Antibodyfragments may be generated by any technique known to one of skill in theart. For example, Fab and F(ab′)₂ fragments may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and the hinge region of the heavy chain.Antibody fragments can be also produced by recombinant DNA technologies.Antibody fragments may be one or more complementarity determiningregions (CDRs) of antibodies, or one or more antigen-binding fragmentsof an antibody.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, camelised antibodies, chimeric antibodies,single-chain Fvs (scFv), single chain antibodies, single domainantibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id antibodies to antibodies of the invention), intrabodies, andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

The term “antibodies or antibody fragments that immunospecifically bindto an IL-9 polypeptide” and analogous terms as used herein refer toantibodies or antibody fragments that specifically bind to an IL-9polypeptide or a fragment of an IL-9 polypeptide and do not specificallybind to other polypeptides. Preferably, antibodies or antibody fragmentsof the invention have a higher affinity to an IL-9 polypeptide or afragment of an IL-9 polypeptide when compared to the affinity to otherpolypeptides or fragments of other polypeptides. The affinity of anantibody is a measure of its bonding with a specific antigen at a singleantigen-antibody site, and is in essence the summation of all theattractive and repulsive forces present in the interaction between theantigen-binding site of an antibody and a particular epitope. Theaffinity of an antibody to a particular antigen (e.g., an IL-9polypeptide or fragment of an IL-9 polypeptide) may be expressed by theequilibrium constant K, defined by the equation K=[Ag Ab]/[Ag][Ab],which is the affinity of the antibody-combining site where [Ag] is theconcentration of free antigen, [Ab] is the concentration of freeantibody and [Ag Ab] is the concentration of the antigen-antibodycomplex. Where the antigen and antibody react strongly together therewill be very little free antigen or free antibody, and hence theequilibrium constant or affinity of the antibody will be high. Highaffinity antibodies are found where there is a good fit between theantigen and the antibody (for a discussion regarding antibody affinity,see Sigal and Ron ed., 1994, Immunology and Inflammation—BasicMechanisms and Clinical Consequences, McGraw-Hill, Inc. New York atpages 56-57; and Seymour et al., 1995, Immunology—An Introduction forthe Health Sciences, McGraw-Hill Book Company, Australia at pages31-32). Preferably, antibodies or antibody fragments thatimmunospecifically bind to an IL-9 polypeptide or fragment thereof donot cross-react with other antigens. That is, antibodies or antibodyfragments of the invention immunospecifically bind to an IL-9polypeptide or fragment thereof with a higher energy than to otherpolypeptides or fragments of other polypeptides (see, e.g., Paul ed.,1989, Fundamental Immunology, 2^(nd) ed., Raven Press, New York at pages332-336 for a discussion regarding antibody specificity). Antibodies orantibody fragments that immunospecifically bind to an IL-9 polypeptidecan be identified, for example, by immunoassays such asradioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),and BIAcore assays (described in Section 5.7, infra) or other techniquesknown to those of skill in the art (see, e.g., Seymour et al., 1995,Immunology—An Introduction for the Health Sciences, McGraw-Hill BookCompany, Australia at pages 33-41 for a discussion of various assays todetermine antibody-antigen interactions in vivo). Preferably antibodiesor antibody fragments that immunospecifically bind to an IL-9polypeptide or fragment thereof only antagonize an IL-9 polypeptide anddo not significantly antagonize other activities.

As used herein, the term “control IgG antibody” refers to an IgGantibody or other “control antibody” that does not immunospecificallybind to an IL-9 polypeptide and preferably does not cross-react with anIL-9 polypeptide.

As used herein, the term “cytokine receptor modulator” refers to anagent that modulates the phosphorylation of a cytokine receptor, theactivation of a signal transduction pathway associated with a cytokinereceptor, and/or the expression of a particular protein such as acytokine. Such an agent may directly or indirectly modulate thephosphorylation of a cytokine receptor, the activation of a signaltransduction pathway associated with a cytokine receptor, and/or theexpression of a particular protein such as a cytokine. Thus, examples ofcytokine receptor modulators include, but are not limited to, cytokines,fragments of cytokines, fusion proteins, and antibodies thatimmunospecifically bind to a cytokine receptor or a fragment of theantibody or cytokine receptor. Further, examples of cytokine receptormodulators include, but are not limited to, peptides, polypeptides(e.g., soluble cytokine receptors), fusion proteins and antibodies thatimmunospecifically binds to a cytokine or a fragment thereof.

As used herein, the term “derivative” in the context of proteinaceousagent (e.g., proteins, polypeptides, peptides, and antibodies) refers toa proteinaceous agent that comprises an amino acid sequence which hasbeen altered by the introduction of amino acid residue substitutions,deletions, and/or additions. The term “derivative” as used herein alsorefers to a proteinaceous agent which has been modified, i.e., by thecovalent attachment of any type of molecule to the proteinaceous agent.For example, but not by way of limitation, an antibody may be modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. A derivative of a proteinaceous agent may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Further, aderivative of a proteinaceous agent may contain one or morenon-classical amino acids. A derivative of a proteinaceous agentpossesses a similar or identical function as the proteinaceous agentfrom which it was derived.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl, nitryl, or amine group. Anorganic molecule may also be esterified, alkylated and/orphosphorylated.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject in which thesubject differs from a healthy, unaffected subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Certain conditions may be characterized as more than onedisorder. For example, certain conditions may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent) which is sufficientto reduce and/or ameliorate the severity and/or duration of a disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, prevent theadvancement of said disease or disorder, cause regression of saiddisease or disorder, prevent the recurrence, development, or onset ofone or more symptoms associated with said disease or disorder, orenhance or improve the prophylactic or therapeutic effect(s) of anothertherapy (e.g., prophylactic or therapeutic agent).

As used herein, the term “epitopes” refers to fragments of a polypeptideor protein having antigenic or immunogenic activity in an animal,preferably in a mammal, and most preferably in a human. An epitopehaving immunogenic activity is a fragment of a polypeptide or proteinthat elicits an antibody response in an animal. An epitope havingantigenic activity is a fragment of a polypeptide or protein to which anantibody immunospecifically binds as determined by any method well-knownto one of skill in the art, for example by immunoassays. Antigenicepitopes need not necessarily be immunogenic.

The term “excipient” as used herein refers to an inert substance whichis commonly used as a diluent, vehicle, preservative, binder orstabilizing agent for drugs which imparts a beneficial physical propertyto a formulation, such as increased protein stability, increased proteinsolubility, and decreased viscosity. Examples of excipients include, butare not limited to, proteins (e.g., serum albumin), amino acids (e.g.,aspartic acid, glutamic acid, lysine, arginine, glycine and histidine),surfactants (e.g., SDS, polysorbate and nonionic surfactant),saccharides (e.g., glucose, sucrose, maltose and trehalose), polyols(e.g., mannitol and sorbitol), fatty acids and phospholipids (e.g.,alkyl sulfonates and caprylate). For additional information regardingexcipients, see Remington's Pharmaceutical Sciences (by Joseph P.Remington, 18^(th) ed., Mack Publishing Co., Easton, Pa.), which isincorporated herein in its entirety.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of asecond, different polypeptide or protein. In another embodiment, afragment of a protein or polypeptide retains at least one function ofthe protein or polypeptide. In another embodiment, a fragment of apolypeptide or protein retains at least two, three, four, or fivefunctions of the polypeptide or protein. Preferably, a fragment of anantibody that immunospecifically binds to an IL-9 polypeptide retainsthe ability to immunospecifically bind to an IL-9 polypeptide. A“functional fragment” is a fragment that retains at least one functionof the protein or polypeptide.

As used herein, the term “fusion protein” refers to a polypeptide orprotein that comprises an amino acid sequence of a first polypeptide orprotein or fragment, analog or derivative thereof, and an amino acidsequence of a heterologous polypeptide or protein (i.e., a secondpolypeptide or protein or fragment, analog or derivative thereofdifferent than the first polypeptide or protein or fragment, analog orderivative thereof). In one embodiment, a fusion protein comprises aprophylactic or therapeutic agent fused to a heterologous protein,polypeptide or peptide. In accordance with this embodiment, theheterologous protein, polypeptide or peptide may or may not be adifferent type of prophylactic or therapeutic agent. For example, twodifferent proteins, polypeptides, or peptides with immunomodulatoryactivity may be fused together to form a fusion protein. In a preferredembodiment, fusion proteins retain or have improved activity relative tothe activity of the original polypeptide or protein prior to being fusedto a heterologous protein, polypeptide, or peptide.

The terms “high concentration” and “concentrated antibody” as usedherein refer to a concentration of 50 mg/ml or higher, preferably 95mg/ml or higher of an antibody (including antibody fragment thereof)that immunospecifically binds to an IL-9 polypeptide, in an antibodyformulation.

As used herein, the term “host cell” includes a particular subject celltransfected or transformed with a nucleic acid molecule and the progenyor potential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transfected with the nucleic acid moleculedue to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome.

As used herein, the terms “human child” or “child” or variations thereofrefer to a human between 24 months of age and 18 years of age.

As used herein, the terms “elderly human,” “elderly,” or variationsthereof refer to a human 65 years old or older, preferably 70 years oldor older.

As used herein, the terms “human infant” or “infant” or variationsthereof refer to a human less than 24 months of age, preferably lessthan 12 months, less than 6 months, less than 3 months, less than 2months, or less than 1 month of age.

As used herein, the terms “human infant born prematurely,” “preterminfant,” or “premature infant,” or variations thereof refer to a humanborn at less than 40 weeks of gestational age, preferably less than 35weeks gestational age, who is less than 6 months old, preferably lessthan 3 months old, more preferably less than 2 months old, and mostpreferably less than 1 month old.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing under whichnucleotide sequences at least 30% (preferably, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) identical to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found, for example, inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and updates), 6.3.1-6.3.6.

Generally, stringent conditions are selected to be about 5 to 110°C.lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (for example, 10 to 50 nucleotides) and at least 60° C. forlong probes (for example, greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents, for example, formamide. For selective or specific hybridization,a positive signal is at least two times background, preferably 10 timesbackground hybridization.

In one, non-limiting example, stringent hybridization conditions arehybridization at 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68° C.In a preferred, non-limiting example stringent hybridization conditionsare hybridization in 6×SSC at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. (i.e., one or more washes at50° C., 55° C., 60° C. or 65° C.).

As used herein, the term “IL-9 polypeptide” refers to IL-9, an analog,derivative or a fragment thereof, including mature and immature forms ofIL-9 (see, Van Snick et al., 1989, J. Exp. Med. 169:363-68 and Yang etal., 1989, Blood 74:1880-84, which are both incorporated by referenceherein in their entireties), or a fusion protein comprising IL-9, ananalog, derivative or a fragment thereof. The IL-9 polypeptide may befrom any species. The nucleotide and/or amino acid sequences of IL-9polypeptides can be found in the literature or public databases, or thenucleotide and/or amino acid sequences can be determined using cloningand sequencing techniques known to one of skill in the art. For example,the nucleotide sequence of human IL-9 can be found in the GenBankdatabase (see, e.g., Accession No. NM_(—)000590; FIG. 12). The aminoacid sequence of human IL-9 can be found in the GenBank database (see,e.g., Accession Nos. A60480, NP_(—)000584 and AAC17735; FIG. 13) and inU.S. Provisional Application No. 60/371,683, entitled, “RecombinantAnti-Interleukin-9 Antibodies,” filed Apr. 12, 2002 (the amino acidsequence of human IL-9 on page 15 is specifically incorporated herein byreference). In a preferred embodiment, an IL-9 polypeptide is humanIL-9, an analog, derivative or a fragment thereof.

As used herein, the terms “IL-9 receptor” and “IL-9R” refer to an IL-9receptor or an analog, derivative, or fragment thereof, or a fusionprotein comprising an IL-9 receptor, an analog, derivative, or afragment thereof. As used herein, the terms “one or more subunits” and“a subunit” in the context of an IL-9R refer to the IL-9Rligand-specific alpha subunit (“IL-9Rα”) and/or common γ_(c) chain (alsopresent in IL-2R, IL-4R, IL-7R, and IL-15R complexes) of the functionalIL-9R or an analog, derivative, or fragment thereof. In a preferredembodiment, a functional IL-9R mediates a proliferative response in Tcells treated with IL-9 as determined by any cell proliferation assayknown to those skilled in the art (e.g., a [³H]-thymidine incorporationassay or a hexosamimidase assay) (see, e.g., Renauld et al., 1992, Proc.Natl. Acad. Sci. USA, 89:5690-94 and Bauer et al., 1998, J. Biol. Chem.273:9255-60, which are both incorporated by reference herein in theirentireties). Preferebly, treating a T cell line expressing a functionalIL-9R (e.g., TS1 RA3 cells (R&D Systems) expressing both human andmurine IL-9Rα) with IL-9, results in a dose-dependent increase in T cellproliferation, as measured by any cell proliferation assay known tothose skilled in the art (see, Renauld et al., 1992, Proc. Natl. Acad.Sci. USA, 89:5690-94 and Bauer et al., 1998, J. Biol. Chem.273:9255-60). In another preferred embodiment, a functional IL-9R,comprising the γ_(c) and IL-9Rα chains, initiates a signaling cascadethrough the Janus kinases JAK1 and JAK3, thereby activating homo- andheterodimers of the signal transducer and activator transcription (STAT)factors STAT-1, STAT-3 and STAT-5 (see, Bauer et al., 1998, J. Biol.Chem. 273:9255-60). In another preferred embodiment, a functional IL-9Rmay prevent apoptosis through a mechanism involving STAT-3 and STAT-5,as determined by apoptosis assays known to those skilled in the art(see, Bauer et al., 1998, J. Biol. Chem. 273:9255-60). The IL-9R or oneor more subunits thereof may be from any species. The nucleotide and/oramino acid sequences of the IL-9R and the subunits thereof can be foundin the literature or in public databases, or the nucleotide and/or aminoacid sequences can be determined using cloning and sequencing techniquesknown to one of skill in the art. For example, the nucleotide sequenceof human IL-9R can be found in the GenBank database (see, e.g.,Accession Nos. NM_(—)002186, NM_(—)176786, and NM_(—)000206; FIG. 14).The amino acid sequence of human IL-9R can be found in the GenBankdatabase (see, e.g., Accession Nos. NP_(—)002177; NP_(—)789743, andNP_(—)000197; FIG. 15) and in U.S. Provisional Application No.60/371,683, entitled, “Recombinant Anti-Interleukin-9 Antibodies,” filedApr. 12, 2002 (the amino acid sequence of human IL-9R on page 16 isherein specifically incorporated by reference). In a preferredembodiment, an IL-9R or one or more subunits thereof is a human IL-9R orone or more subunits thereof, an analog, derivative, or a fragmentthereof.

As used herein, the term “immunomodulatory agent” and variations thereofincluding, but not limited to, immunomodulatory agents, immunomodulantsor immunomodulatory drugs, refer to an agent that modulates a host'simmune system. In a specific embodiment, an immunomodulatory agent is anagent that shifts one aspect of a subject's immune response. In certainembodiments, an immunomodulatory agent is an agent that inhibits orreduces a subject's immune system (i.e., an immunosuppressant agent). Incertain other embodiments, an immunomodulatory agent is an agent thatactivates or increases a subject's immune system (i.e., animmunostimulatory agent). In accordance with the invention, animmunomodulatory agent used in the combination therapies of theinvention does not include an antibody of the invention.Immunomodulatory agents include, but are not limited to, smallmolecules, peptides, polypeptides, proteins, nucleic acids (e.g., DNAand RNA nucleotides including, but not limited to, antisense nucleotidesequences, triple helices, RNAi, and nucleotide sequences encodingbiologically active proteins, polypeptides or peptides), antibodies,synthetic or natural inorganic molecules, mimetic agents, and syntheticor natural organic molecules. The term “in combination” as used hereinrefers to the use of more than one therapies (e.g., prophylactic and/ortherapeutic agents). The use of the term “in combination” does notrestrict the order in which therapies (e.g., prophylactic and/ortherapeutic agents) are administered to a subject with a disease ordisorder (e.g., a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof). A first therapy (e.g., a prophylactic or therapeuticagent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,or 12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disease ordisorder (e.g., disease or disorder associated with or characterized byaberrant expression and/or activity of an IL-9 polypeptide, a disease ordisorder associated with or characterized by aberrant expression and/oractivity of the IL-9R or one or more subunits thereof, an autoimmunedisease, an inflammatory disease, a proliferative disease, or aninfection (preferably, a respiratory infection), or one or more symptomsthereof).

As used herein, the term “immunospecifically binds to an antigen” andanalogous terms refer to peptides, polypeptides, proteins, fusionproteins and antibodies (including antibody fragments thereof) thatspecifically bind to an antigen or a fragment and do not specificallybind to other antigens. A peptide, polypeptide, protein, or antibodythat immunospecifically binds to an antigen may bind to other peptides,polypeptides, or proteins with lower affinity as determined by, e.g.,immunoassays, BIAcore, or other assays known in the art. Antibodies(including antibody fragments thereof) that immunospecifically bind toan antigen may be cross-reactive with related antigens. Preferably,antibodies (including antibody fragments thereof) thatimmunospecifically bind to an antigen do not significantly cross-reactwith other antigens (i.e., is not detectable in routine immunologicalassays). An antibody binds specifically to an antigen when it binds tothe antigen with higher affinity than to any cross-reactive antigen asdetermined using experimental techniques, such as radioimmunoassays(RIAs) and enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul,ed., 1989, Fundamental Immunology, 2nd ed., Raven Press, New York atpages 332-336 for a discussion regarding antibody specificity.

As used herein, the term “in combination” refers to the use of more thanone therapy (e.g., more than one prophylactic agent and/or therapeuticagent). The use of the term “in combination” does not restrict the orderin which therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a respiratory condition. A first therapy(e.g., a first prophylactic or therapeutic agent) can be administeredprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapy (e.g., a second prophylactic ortherapeutic agent) to a subject with a respiratory condition.

The term “inorganic salt” as used herein refers to any compoundscontaining no carbon that result from replacement of part or all of theacid hydrogen or an acid by a metal or a group acting like a metal andare often used as tonicity adjusting compounds in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄, etc.

As used herein, the term “isolated” in the context of an organic orinorganic molecule (whether it be a small or large molecule), other thana proteinaceous agent or nucleic acid molecule, refers to an organic orinorganic molecule substantially free of a different organic orinorganic molecule. Preferably, an organic or inorganic molecule is 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free of a second, differentorganic or inorganic molecule. In a preferred embodiment, an organicand/or inorganic molecule is isolated.

As used herein, the term “isolated” in the context of a proteinaceousagent (e.g., a peptide, polypeptide, fusion protein, or antibody) refersto a proteinaceous agent which is substantially free of cellularmaterial or contaminating proteins from the cell or tissue source fromwhich it is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of a proteinaceousagent in which the proteinaceous agent is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, a proteinaceous agent that is substantially free ofcellular material includes preparations of a proteinaceous agent havingless than about 30%, 20%, 10%, or 5% (by dry weight) of heterologousprotein, polypeptide, peptide, or antibody (also referred to as a“contaminating protein”). When the proteinaceous agent is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the proteinaceous agent preparation. When the proteinaceousagent is produced by chemical synthesis, it is preferably substantiallyfree of chemical precursors or other chemicals, i.e., it is separatedfrom chemical precursors or other chemicals which are involved in thesynthesis of the proteinaceous agent. Accordingly, such preparations ofa proteinaceous agent have less than about 30%, 20%, 10%, 5% (by dryweight) of chemical precursors or compounds other than the proteinaceousagent of interest. In a specific embodiment, proteinaceous agentsdisclosed herein are isolated. In a preferred embodiment, an antibody ofthe invention is isolated. In a specific embodiment, an “isolated”antibody is purified by a multi-step purification process that comprisesthree chromatography steps (cation exchange, protein A and anionexchange), a nanofiltration step, and a low pH treatment step (for adetailed description, see Section 6, infra).

As used herein, the term “isolated” in the context of nucleic acidmolecules refers to a nucleic acid molecule which is separated fromother nucleic acid molecules which are present in the natural source ofthe nucleic acid molecule. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. In a specific embodiment, nucleicacid molecules are isolated; however, “isolated” excludes members of apopulation of a library of clones such as a cDNA library.

The phrase “low to undetectable levels of aggregation” as used hereinrefers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1% and most preferably no morethan 0.5% aggregation by weight of protein as measured by highperformance size exclusion chromatography (HPSEC).

The term “low to undetectable levels of fragmentation” as used hereinrefers to samples containing equal to or more than 80%, 85%, 90%, 95%,98% or 99% of the total protein, for example, in a single peak asdetermined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (oras many peaks as there are subunits) by reduced Capillary GelElectrophoresis (rCGE), representing the non-degraded antibody or anon-degraded fragment thereof, and containing no other single peakshaving more than 5%, more than 4%, more than 3%, more than 2%, more than1%, or more than 0.5% of the total protein in each. The term “reducedCapillary Gel Electrophoresis” as used herein refers to capillary gelelectrophoresis under reducing conditions sufficient to reduce disulfidebonds in an antibody.

As used herein, the terms “manage,” “managing,” and “management” referto the beneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent), which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore therapies (e.g., one or more prophylactic or therapeutic agents) to“manage” a disease so as to prevent the progression or worsening of thedisease.

As used herein, the term “mast cell modulator” refers to an agent whichmodulates the activation of a mast cell, mast cell degranulation, and/orexpression of a particular protein such as a cytokine. Such an agent maydirectly or indirectly modulate the activation of a mast cell,degranulation of the mast cell, and/or the expression of a particularprotein such as a cytokine. Non-limiting examples of mast cellmodulators include, but are not limited to, small molecules, peptides,polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotidesincluding, but not limited to, antisense nucleotide sequences, triplehelices, RNAi, and nucleotide sequences encoding biologically activeproteins, polypeptides, or peptides), fusion proteins, antibodies,synthetic or natural inorganic molecules, synthetic or natural organicmolecule, or mimetic agents which inhibit and/or reduce the expression,function, and/or activity of a stem cell factor, a mast cell protease, acytokine (such as IL-3, IL-4, and IL-9), a cytokine receptor (such asIL-3R, IL-4R, and IL-9R), and a stem cell receptor. Other non-limitingexamples of mast cell modulators include, but are not limited to smallmolecules, peptides, polypeptides, proteins, nucleic acids (e.g., DNAand RNA nucleotides including, but not limited to, antisense nucleotidesequences, triple helices, RNAi, and nucleotide sequences encodingbiologically active proteins, polypeptides, or peptides), fusionproteins, antibodies, synthetic or natural inorganic molecules,synthetic or natural organic molecule, or mimetic agents which inhibitand/or reduce the expression, function and/or activity of IgE. Incertain embodiments, a mast cell modulator is an agent that prevents orreduces the activation of additional mast cells following degranulationof mast cells. In other embodiments, a mast cell modulator is an agentthat inhibits or reduces mast cell degranulation.

As used herein, the terms “non-responsive” and refractory” describepatients treated with a currently available therapy (e.g., prophylacticor therapeutic agent) for a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof which is not clinically adequate to relieve oneor more symptoms associated with the disorder. Typically, such patientssuffer from severe, persistently active disease and require additionaltherapy to ameliorate the symptoms associated with the disorder.

The phrase “pharmaceutically acceptable” as used herein means approvedby a regulatory agency of the Federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

The term “polyol” as used herein refers to a sugar that contains many—OH groups compared to a normal saccharide.

As used herein, the terms “prevent,” “preventing,” and “prevention”refer to the inhibition of the development or onset of disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, or theprevention of the recurrence, onset, or development of one or moresymptoms of a respiratory condition in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or the administration of a combination of therapies (e.g., a combinationof prophylactic or therapeutic agents).

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of the onset,recurrence or development of a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof. In certain embodiments, the term “prophylacticagent” refers to an antibody that immunospecifically binds to an IL-9polypeptide. In certain other embodiments, the term “prophylactic agent”refers to an agent other than an antibody that immunospecifically bindsto an IL-9 polypeptide. Preferably, a prophylactic agent is an agentwhich is known to be useful to or has been or is currently being used tothe prevent or impede the onset, development, progression and/orseverity of a disease or disorder associated with or characterized byaberrant expression and/or activity of an IL-9 polypeptide, a disease ordisorder associated with or characterized by aberrant expression and/oractivity of the IL-9R or one or more subunits thereof, an autoimmunedisease, an inflammatory disease, a proliferative disease, or aninfection (preferably, a respiratory infection), or one or more symptomsthereof. Prophylactic agents may be characterized as different agentsbased upon one or more effects that the agents have in vitro and/or invivo. For example, a mast cell modulator may also be characterized as animmunomodulatory agent.

As used herein, the term “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention of the development, recurrence, or onset ofa disease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof,or to enhance or improve the prophylactic effect(s) of another therapy(e.g., a prophylactic agent).

As used herein, a “prophylactic protocol” refers to a regimen for dosingand timing the administration of one or more therapies (e.g., one ormore prophylactic agents) that has a prophylactic effect.

A used herein, a “protocol” includes dosing schedules and dosingregimens. The protocols herein are methods of use and includeprophylactic and therapeutic protocols.

The term “saccharide” as used herein refers to a class of molecules thatare derivatives of polyhydric alcohols. Saccharides are commonlyreferred to as carbohydrates and may contain different amounts of sugar(saccharide) units, e.g., monosaccharides, disaccharides andpolysaccharides.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Side effects arealways unwanted, but unwanted effects are not necessarily adverse. Anadverse effect from a therapy (e.g., a prophylactic or therapeuticagent) might be harmful, uncomfortable, or risky.

Side effects from administration of REMICADE™ include, but are notlimited to, risk of serious infection and hypersensitivity reactions.Other side effects range from nonspecific symptoms such as fever orchills, pruritus or urticaria, and cardiopulmonary reactions such aschest pain, hypotension, hypertension or dyspnea, to effects such asmyalgia and/or arthralgia, rash, facial, hand or lip edema, dysphagia,sore throat, and headache. Yet other side effects include, but are notlimited to, abdominal hernia, splenic infarction, splenomegaly,dizziness, upper motor neuron lesions, lupus erythematosus syndrome,rheumatoid nodules, ceruminosis, abdominal pain, diarrhea, gastriculcers, intestinal obstruction, intestinal perforation, intestinalstenosis, nausea, pancreatitis, vomiting, back pain, bone fracture,tendon disorder or injury, cardiac failure, myocardial ischema,lymphoma, thrombocytopenia, cellulitis, anxiety, confusion, delirium,depression, somnolence, suicide attempts, anemia, abscess, bacterialinfections, and sepsis. Side effects from administration of ENBREL™include, but are not limited to, risk of serious infection and sepsis,including fatalities. Adverse side effects range from serious infectionssuch as pyelonephritis, bronchitis, septic arthritis, abdominal abscess,cellulitis, osteomyelitis, wound infection, pneumonia, foot abscess, legulcer, diarrhea, sinusitis, sepsis, headache, nausea, rhinitis,dizziness, pharyngitis, cough, asthenia, abdominal pain, rash,peripheral edema, respirator disorder, dyspepsia, sinusitis, vomiting,mouth ulcer, alopecia, and pheumonitis to other less frequent adverseeffects such as heart failure, myocardial infarction, myocardiaischemia, cerebral ischemia, hyertension, hypotension, cholcystitis,pancreatitis, gastrointestinal hemorrhage, bursitis, depression,dyspnea, deep vein thrombosis, pulmonary embolism, membranousglomerulonephropathy, polymyositis, and thrombophlebitis. The sideeffects resulting from administration of methotrexate include, but arenot limited to, serious toxic reactions, which can be fatal, such asunexpectedly severe bone marrow suppression, gastrointestinal toxicity,hepatotoxicity, fibrosis and cirrhosis after prolonged use, lungdiseases, diarrhea and ulcerative stomatitis, malignant lymphomas andoccasionally fatal severe skin reactions.

Side effects from chemotherapy include, but are not limited to,gastrointestinal toxicity such as, but not limited to, early andlate-forming diarrhea and flatulence; nausea; vomiting; anorexia;leukopenia; anemia; neutropenia; asthenia; abdominal cramping; fever;pain; loss of body weight; dehydration; alopecia; dyspnea; insomnia;dizziness, mucositis, xerostomia, and kidney failure, as well asconstipation, nerve and muscle effects, temporary or permanent damage tokidneys and bladder, flu-like symptoms, fluid retention, and temporaryor permanent infertility. Side effects from radiation therapy includebut are not limited to fatigue, dry mouth, and loss of appetite. Otherside effects include gastrointestinal toxicity such as, but not limitedto, early and late-forming diarrhea and flatulence; nausea; vomiting;anorexia; leukopenia; anemia; neutropenia; asthenia; abdominal cramping;fever; pain; loss of body weight; dehydration; alopecia; dyspnea;insomnia; dizziness, mucositis, xerostomia, and kidney failure. Sideeffects from biological therapies/immunotherapies include but are notlimited to rashes or swellings at the site of administration, flu-likesymptoms such as fever, chills and fatigue, digestive tract problems andallergic reactions. Side effects from hormonal therapies include but arenot limited to nausea, fertility problems, depression, loss of appetite,eye problems, headache, and weight fluctuation. Additional undesiredeffects typically experienced by patients are numerous and known in theart. Many are described in the Physicians' Desk Reference (56th ed.,2002 and 57th ed., 2003).

As used herein, the term “small molecules” and analogous terms include,but are not limited to, peptides, peptidomimetics, amino acids, aminoacid analogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such agents.

The terms “stability” and “stable” as used herein in the context of aliquid formulation comprising an antibody (including antibody fragmentthereof) that immunospecifically binds to an IL-9 polypeptide refer tothe resistance of the antibody or (including antibody fragment thereof)in the formulation to degradation or fragmentation under givenmanufacture, preparation, transportation and storage conditions. The“stable” formulations of the invention retain biological activity undergiven manufacture, preparation, transportation and storage conditions.The stability of said antibody (including antibody fragment thereof) canbe assessed by degrees of degradation or fragmentation, as measured byHPSEC, compared to a reference formulation. For example, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an antibody (including antibody fragment thereof) (e.g.,4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4) in histidine-HCl buffer, pH 6.0that contains 150 mM NaCl, which reference formulation regularly gives asingle monomer peak (≧97% area) by HPSEC. Alternatively, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an antibody (including antibody fragment thereof) (e.g.,4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4) in histidine-HCl buffer at pH6.0, which reference formulation regularly gives a single monomer peak(≧97% area) by HPSEC. The overall stability of a formulation comprisingan antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide can be assessed byvarious immunological assays including, for example, ELISA andradioimmunoassay using isolated an IL-9 polypeptide molecules or cellsexpressing the same.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., amonkey, such as a cynomolgous monkey, chimpanzee, and a human), and morepreferably a human. In a certain embodiment, the subject is a mammal,preferably a human, with a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof. In another embodiment, the subject is a farmanimal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat) with adisease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof.In another embodiment, the subject is a mammal, preferably a human, atrisk of developing a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof (e.g., an immunocompromised or immunosuppressedmammal). In another embodiment, the subject is not an immunocompromisedor immunosuppressed mammal, preferably a human. In another embodiment,the subject is a mammal, preferably a human, with a lymphocyte countthat is not under approximately 500 cells/mm³. In another embodiment,the subject is a human infant or a human infant born prematurely. Inanother embodiment, the subject is a human child or a human adult. Inanother embodiment, the subject is a human child with bronchopulmonarydysplasia, congenital heart diseases, or cystic fibrosis. In anotherembodiment, the subject is an elderly human. In yet another embodiment,the subject is a human in an institution or group home, such as, but notlimited to, a nursing home.

The term “substantially free of surfactant” as used herein refers to aformulation of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide, said formulationcontaining less than 0.0005%, less than 0.0003%, or less than 0.0001% ofsurfactants.

The term “substantially free of sugars, sugar alcohols, and amino acidsother than histidine” as used herein refers to a formulation of anantibody (including antibody fragment thereof) that immunospecificallybinds to an IL-9 polypeptide, said formulation containing less than0.0005%, less than 0.0003%, or less than 0.0001% of sugars, sugaralcohols, and amino acids other than histidine.

As used herein, the term “synergistic” refers to a combination oftherapies (e.g., prophylactic or therapeutic agents) which is moreeffective than the additive effects of any two or more single therapies(e.g., one or more prophylactic or therapeutic agents). A synergisticeffect of a combination of therapies (e.g., a combination ofprophylactic or therapeutic agents) permits the use of lower dosages ofone or more of therapies (e.g., one or more prophylactic or therapeuticagents) and/or less frequent administration of said therapies to asubject with a respiratory condition. The ability to utilize lowerdosages of therapies (e.g., prophylactic or therapeutic agents) and/orto administer said therapies less frequently reduces the toxicityassociated with the administration of said therapies to a subjectwithout reducing the efficacy of said therapies in the prevention ortreatment of a respiratory condition. In addition, a synergistic effectcan result in improved efficacy of therapies (e.g., prophylactic ortherapeutic agents) in the prevention or treatment of a respiratorycondition. Finally, the synergistic effect of a combination of therapies(e.g., prophylactic or therapeutic agents) may avoid or reduce adverseor unwanted side effects associated with the use of any single therapy.

As used herein, the term “T cell receptor modulator” refers to an agentwhich modulates the phosphorylation of a T cell receptor, the activationof a signal transduction pathway associated with a T cell receptorand/or the expression of a particular protein associated with T cellreceptor activity such as a cytokine. Such an agent may directly orindirectly modulate the phosphorylation of a T cell receptor, theactivation of a signal transduction pathway associated with a T cellreceptor, and/or the expression of a particular protein associated withT cell receptor activity such as a cytokine. Examples of T cell receptormodulators include, but are not limited to, peptides, polypeptides,proteins, fusion proteins and antibodies which immunospecifically bindto a T cell receptor or a fragment thereof. Further, examples of T cellreceptor modulators include, but are not limited to, proteins, peptides,polypeptides (e.g., soluble T cell receptors), fusion proteins andantibodies that immunospecifically bind to a ligand for a T cellreceptor or fragments thereof.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the prevention, treatment,management, or amelioration of a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof. In certain embodiments, the term “therapeuticagent” refers to an antibody that binds to an IL-9 polypeptide. Incertain other embodiments, the term “therapeutic agent” refers an agentother than an antibody that immunospecifically binds to an IL-9polypeptide. Preferably, a therapeutic agent is an agent that is knownto be useful for, or has been or is currently being used for theprevention, treatment, management, or amelioration of a disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof. Therapeuticagents may be characterized as different agents based upon one or moreeffects the agents have in vivo and/or in vitro, for example, ananti-inflammatory agent may also be characterized as an immunomodulatoryagent.

As used herein, the term “therapeutically effective amount” refers tothe amount of a therapy (e.g., an antibody that immunospecifically bindsto an IL-9 polypeptide), that is sufficient to reduce the severity of adisease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof,reduce the duration of a respiratory condition, ameliorate one or moresymptoms of a disease or disorder associated with or characterized byaberrant expression and/or activity of an IL-9 polypeptide, a disease ordisorder associated with or characterized by aberrant expression and/oractivity of the IL-9R or one or more subunits thereof, an autoimmunedisease, an inflammatory disease, a proliferative disease, or aninfection (preferably, a respiratory infection), or one or more symptomsthereof, autoimmune diseases, inflammatory diseases, proliferativediseases, or infections (preferably, respiratory infections), or one ormore symptoms thereof, cause regression of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, or enhance orimprove the therapeutic effect(s) of another therapy.

The terms “therapies” and “therapy” can refer to any protocol(s),method(s), and/or agent(s) that can be used in the prevention,treatment, management, or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof. In certainembodiments, the terms “therapy” and “therapy” refer to anti-viraltherapy, anti-bacterial therapy, anti-fungal therapy, biologicaltherapy, supportive therapy, and/or other therapies useful in treatment,management, prevention, or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof known to skilledmedical personnel.

As used herein, the term “therapeutic protocol” refers to a regimen fordosing and timing the administration of one or more therapies (e.g.,therapeutic agents) that has a therapeutic effective.

As used herein, the terms “treat,” “treatment,” and “treating” refer tothe reduction or amelioration of the progression, severity, and/orduration of a disease or disorder associated with or characterized byaberrant expression and/or activity of an IL-9 polypeptide, a disease ordisorder associated with or characterized by aberrant expression and/oractivity of the IL-9R or one or more subunits thereof, an autoimmunedisease, an inflammatory disease, a proliferative disease, or aninfection (preferably, a respiratory infection), or one or more symptomsthereof resulting from the administration of one or more therapies(including, but not limited to, the administration of one or moreprophylactic or therapeutic agents). In certain embodiments, such termsrefer to a reduction in the swelling of organs or tissues, or areduction in the pain associated with a respiratory condition. In otherembodiments, such terms refer to a reduction in the inflammation orconstriction of an airway(s) associated with asthma. In otherembodiments, such terms refer to a reduction in the replication of aninfectious agent, or a reduction in the spread of an infectious agent toother organs or tissues in a subject or to other subjects. In otherembodiments, such terms refer to the reduction of the release ofinflammatory agents by mast cells, or the reduction of the biologicaleffect of such inflammatory agents. In other embodiments, such termsrefer to a reduction of the growth, formation and/or increase in thenumber of hyperproliferative cells (e.g., cancerous cells). In yet otherembodiments, such terms refer to the eradication, removal or control ofprimary, regional or metastatic cancer (e.g., the minimization or delayof the spread of cancer).

The term “very little to no loss of the biological activities” as usedherein refers to antibody activities, including but not limited to,specific binding abilities of antibodies (including antibody fragmentsthereof) to an IL-9 polypeptide as measured by various immunologicalassays, including, but not limited to ELISAs and radioimmunoassays. Inone embodiment, the antibodies (including antibody fragments thereof) ofthe formulations of the invention retain approximately 50%, preferably55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of the ability toimmunospecifically bind to an IL-9 polypeptide as compared to areference antibody (including antibody fragment thereof) (e.g., 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4) as measured by an immunological assay knownto one of skill in the art or described herein. For example, an ELISAbased assay may be used to compare the ability of an antibody (includingantibody fragment thereof) to immunospecifically bind to an IL-9polypeptide to a 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10,7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 reference standard. Inthis assay, referred to as the IL-9 Binding ELISA, plates are coatedwith an isolated IL-9 and the binding signal of a set concentration of a4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 reference standard is compared tothe binding signal of the same concentration of a test antibody(including antibody fragment thereof). A “reference standard” as usedherein refers to an antibody (including antibody fragment thereof)(e.g., 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4) that is frozen at −70° C.consisting of 10 mg/ml of an antibody (including antibody fragmentthereof) (e.g., 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10,7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4) in histidine-HClbuffer, pH 6.0, and containing 150 mM NaCl, which reference formulationregularly gives a single monomer peak (≧97% area) by HPSEC. In anotherembodiment, the term “very little to no loss of biological activities”as used herein refers to antibody activities, including other effectoractivities of the antibody.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 7) of 4D4 with the VH CDR1 (SEQ ID NO.: 1), the VHCDR2 (SEQ ID NO.: 2), and the VH CDR3 (SEQ ID NO.: 3) underlined,starting in order from VH CDR1 at the far left; and (B) variable lightdomain (SEQ ID. NO.: 8) of 4D4, with the VL CDR1 (SEQ ID NO.: 4), the VLCDR2 (SEQ ID NO.: 5), and the VL CDR3 (SEQ ID NO.: 6) underlined,starting in order from VL CDR1 at the far left.

FIGS. 2A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 9) of 4D4H2-1 D11, with the VH CDR1 (SEQ ID NO.: 1),the VH CDR2 (SEQ ID NO.: 10), and the VH CDR3 (SEQ ID NO.: 3)underlined, starting in order from VH CDR1 at the far left; and (B)variable light domain (SEQ ID. NO.: 8) of 4D4H2-1 D11, the VL CDR1 (SEQID NO.: 4), the VL CDR2 (SEQ ID NO.: 5), and the VL CDR3 (SEQ ID NO.: 6)underlined, starting in order from VL CDR1 at the far left.

FIGS. 3A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 15) of 4D4com-XF-9, with the VH CDR1 (SEQ ID NO.:1), the VH CDR2 (SEQ ID NO.: 10), and the VH CDR3 (SEQ ID NO.: 12)underlined, starting in order from VH CDR1 at the far left; and (B)variable light domain (SEQ ID. NO.: 16) of 4D4com-XF-9, the VL CDR1 (SEQID NO.: 13), the VL CDR2 (SEQ ID NO.: 14), and the VL CDR3 (SEQ ID NO.:64) underlined, starting in order from VL CDR1 at the far left.

FIGS. 4A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 17) of 4D4com-2F9, with the VH CDR1 (SEQ ID NO.: 1),the VH CDR2 (SEQ ID NO.: 10), and the VH CDR3 (SEQ ID NO.: 12)underlined, starting in order from VH CDR1 at the far left; and (B)variable light domain (SEQ ID. NO.: 18) of 4D4com-2F9, with the VL CDR1(SEQ ID NO.: 4), the VL CDR2 (SEQ ID NO.: 14), and the VL CDR3 (SEQ IDNO.: 65) underlined, starting in order from VL CDR1 at the far left.

FIGS. 5A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 21) of 7F3, with the VH CDR1 (SEQ ID NO.: 19), theVH CDR2 (SEQ ID NO.: 2), and the VH CDR3 (SEQ ID NO.: 3) underlined,starting in order from VH CDR1 at the far left; and (B) variable lightdomain (SEQ ID. NO.: 22) of 7F3, with the VL CDR1 (SEQ ID NO.: 4), theVL CDR2 (SEQ ID NO.: 5), and the VL CDR3 (SEQ ID NO.: 20) underlined,starting in order from VL CDR1 at the far left.

FIGS. 6A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 23) of 71A10, with the VH CDR1 (SEQ ID NO.: 19), theVH CDR2 (SEQ ID NO.: 61), and the VH CDR3 (SEQ ID NO.: 3) underlined,starting in order from VH CDR1 at the far left; and (B) variable lightdomain (SEQ ID. NO.: 24) of 71A10, the VL CDR1 (SEQ ID NO.: 4), the VLCDR2 (SEQ ID NO.: 5), and the VL CDR3 (SEQ ID NO.: 20) underlined,starting in order from VL CDR1 at the far left.

FIGS. 7A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 21) of 7F3 22D3, with the VH CDR1 (SEQ ID NO.: 19),the VH CDR2 (SEQ ID NO.: 2), and the VH CDR3 (SEQ ID NO.: 3) underlined,starting in order from VH CDR1 at the far left; and (B) variable lightdomain (SEQ ID. NO.: 25) of 7F3 22D3, with the VL CDR1 (SEQ ID NO.: 4),the VL CDR2 (SEQ ID NO.: 14), and the VL CDR3 (SEQ ID NO.: 20)underlined, starting in order from VL CDR1 at the far left.

FIGS. 8A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 27) of 7F3com-2H2, the VH CDR1 (SEQ ID NO.: 26),with the VH CDR2 (SEQ ID NO.: 61), and the VH CDR3 (SEQ ID NO.: 3) areunderlined, starting in order from VH CDR1 at the far left; and (B)variable light domain (SEQ ID. NO.: 28) of 7F3com-2H2, the VL CDR1 (SEQID NO.: 62), the VL CDR2 (SEQ ID NO.: 63), and the VL CDR3 (SEQ ID NO.:20) underlined, starting in order from VL CDR1 at the far left.

FIGS. 9A-B show the nucleotide sequences of the (A) variable heavydomain (SEQ ID NO.: 43) of 7F3com-2H2 with the VH CDR1 (SEQ ID NO.: 44),the VH CDR2 (SEQ ID NO.: 45) and the VH CDR3 (SEQ ID NO.: 46)underlined, starting in order from VH CDR1 at the far left; and (B)variable light domain (SEQ ID NO.: 47) of 7F3com-2H2 with the VL CDR1(SEQ ID NO.: 48), the VL CDR2 (SEQ ID NO.:49), and the VL CDR3 (SEQ IDNO.: 50) underlined, starting in order from VL CDR1 at the far left.

FIGS. 10A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 29) of 7F3com-3H5, with the VH CDR1 (SEQ ID NO.:19), the VH CDR2 (SEQ ID NO.: 61), and the VH CDR3 (SEQ ID NO.: 3)underlined, starting in order from VH CDR1 at the far left and (B)variable light domain (SEQ ID. NO.: 30) of 7F3com-3H5, with the VL CDR1(SEQ ID NO.: 4), the VL CDR2 (SEQ ID NO.: 14), and the VL CDR3 (SEQ IDNO.: 20) underlined, starting in order from VL CDR1 at the far left.

FIGS. 11A-B show the amino acid sequences of the (A) variable heavydomain (SEQ ID NO.: 31) of 7F3com-3D4, with the VH CDR1 (SEQ ID NO.:26), the VH CDR2 (SEQ ID NO.: 61), and the VH CDR3 (SEQ ID NO.: 3)underlined, starting in order from VH CDR1 at the far left and (B)variable light domain (SEQ ID. NO.: 32) of 7F3com-3D4, with the VL CDR1(SEQ ID NO.: 62), the VL CDR2 (SEQ ID NO.: 14), and the VL CDR3 (SEQ IDNO.: 20) underlined, starting in order from VL CDR1 at the far left.

FIG. 12 shows the nucleotide sequence of human IL-9 (SEQ ID NO.: 51)located in the GenBank database (Accession No. NM_(—)000590).

FIGS. 13A-C show the amino acid sequences of human IL-9 located in theGenBank database: (A) Accession No. A60480 (SEQ ID NO.: 52); (B)Accession No. NP_(—)000584 (SEQ ID NO.: 53); and (C)AAC17735 (SEQ IDNO.: 54).

FIGS. 14A-C show the nucleotide sequences of human IL-9R subunits foundin the GenBank database. (A) Accession No. NM_(—)002186 (SEQ ID NO.: 55)and (B) Accession No. NM_(—)176786 (SEQ ID NO.: 56) are the nucleotidesequences of the human IL-9R alpha subunit isoform precursors. (C)Accession No. NM_(—)000206 (SEQ ID NO.: 57) is the nucleotide sequenceof the human IL-9R gamma chain.

FIGS. 15A-C show the amino acid sequences of human IL-9R found in theGenBank database. (A) Accession No. NP_(—)002177 (SEQ ID NO.: 58) and(B) Accession No. NP_(—)789743 (SEQ ID NO.: 59) are the amino acidsequences of the human IL-9R alpha subunit isoform precursors; and (C)Accession No. NP_(—)000197 (SEQ ID NO: 60) is the amino acid sequence ofthe human IL-9R gamma chain.

FIG. 16 is a schematic diagram showing the outline for preparingpurified antibodies that immunospecifically bind to an IL-9 polypeptide.

5. DETAILED DESCRIPTION OF THE INVENTION

The liquid formulations of the present invention provide a ready-to-usepreparation of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide for administering to asubject without having to reconstitute the preparation accurate andaseptical techniques, and waiting for a period of time until thesolution clarifies before administering the formulation to the subject.In addition, such reconstituted solutions must be used within a certainperiod, leading to very costly waste. It simplifies the procedure ofadministering the formulation to a subject for a healthcareprofessional. Furthermore, due to its high stability during the storage,the formulations of the present invention can contain an antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide at concentrations in the range of about 15 mg/ml toabout 300 mg/ml without causing an adverse effect on the biologicalactivities of the antibody due to protein aggregation and/orfragmentation during a prolonged storage. Such stability not onlyensures the efficacy of the antibodies but also reduces possible risksof adverse effects in a subject. Furthermore, the use of fewercomponents in the formulation reduces the risk of contamination. Inaddition, the manufacturing process of the liquid formulations of thepresent invention is simplified and more efficient than themanufacturing process for the lyophilized version because all stages ofthe manufacturing of the liquid formulations are carried out in anaqueous solution, involving no drying process, such as lyophilizationand freeze-drying. Accordingly, it is more cost effective as well.

5.1. Antibody Formulations

The liquid formulations of the present invention provide antibodyformulations which are substantially free of surfactants, and/or otherexcipients such as sugars, sugar alcohols, amino acids (preferably, witha pKa value of less than 5 or higher than 7 and/or amino acids otherthan histidine or lysine), and yet exhibit high stability during longperiods of storage. In a specific embodiment, such antibody formulationsare homogeneous. In a preferred embodiment, the formulations of theinvention are sterile. The formulations of the present inventioncomprise an aqueous carrier, histidine and NaCl buffer, and an antibody(including antibody fragment thereof) which immunospecifically binds toan IL-9 polypeptide at concentrations of about 15 mg/ml to about 300mg/ml. In one embodiment, the formulations of the invention do notcomprise other ingredients except for water or suitable solvents. Inanother preferred embodiment, the water is distilled. In a specificembodiment, the antibody that immunospecifically binds to an IL-9polypeptide which is included in the liquid formulations of theinvention is 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or an antigen-bindingfragment thereof. In another embodiment, the antibody thatimmunospecifically binds to an IL-9 polypeptide which is included in theliquid formulations of the invention is not 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or an antigen-binding fragment thereof. In a preferredembodiment, the antibody that immunospecifically binds to an IL-9polypeptide which is included in the liquid formulation of the inventionis an antibody (including antibody fragment thereof) comprising one ormore of the VH CDRs and/or one or more of the VL CDRs listed in Table 1,supra. In another embodiment, the antibody (including antibody fragmentthereof) that immunospecifically binds to an IL-9 polypeptide which isincluded in the liquid formulations of the invention is an antibody(including antibody fragment thereof) conjugated to another moiety,including but not limited to, a heterologous polypeptide, anotherantibody (including antibody fragment thereof), a marker sequence, adiagnostic agent, a therapeutic agent, a radioactive metal ion, apolymer, albumin, and a solid support. In yet another embodiment, liquidformulations of the invention comprise two or more antibodies or(including antibody fragments thereof) that immunospecifically binds toan IL-9 polypeptide, wherein at least one of the antibodies (includingantibody fragments thereof) is 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4or an antigen-binding fragment thereof.

The concentration of an antibody (including antibody fragment thereof)that immunospecifically binds to an IL-9 polypeptide which is includedin the liquid formulations of the invention is at least 15 mg/ml, atleast 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml,at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least 55mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml, atleast 95 mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, atleast 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least 150mg/ml, at least 175 mg/ml, at least 200 mg/ml, at least 250 mg/ml, atleast 275 mg/ml, or at least 300 mg/ml. In a specific embodiment, theconcentration of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide which is included in theliquid formulation of the invention is about 75 mg/ml, about 100 mg/ml,about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml,about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml.In another embodiment, the concentration of an antibody (includingantibody fragment thereof) that immunospecifically binds to an IL-9polypeptide which is included in the liquid formulation of the inventionis between 15-500 mg/ml, between 50-300 mg/ml, between 50-250 mg/ml,between 50-200 mg/ml, between 50-175 mg/ml, between 50-150 mg/ml,between 50-125 mg/ml, or between 50-100 mg/ml.

Preferably, the formulation is buffered by histidine (although otherappropriate buffers may be used). The concentration of histidine whichis included in the liquid formulations of the invention ranges from 1 mMto 100 mM, preferably 5 mM to 50 mM, and more preferably 10 mM to about25 mM. In a specific embodiment, the concentration of histidine which isincluded in the liquid formulations of the invention is 5 mM, 10 mM, 15mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. Histidine can bein the form of L-histidine, D-histidine, or a mixture thereof, butL-histidine is the most preferable. Histidine can be also in the form ofhydrates. Histidine may be used in a form of pharmaceutically acceptablesalt, such as hydrochloride (e.g., monohydrochloride anddihydrochloride), hydrobromide, sulfate, acetate, etc. The purity ofhistidine should be at least 98%, preferably at least 99%, and mostpreferably at least 99.5%. As used herein, the term “purity” in thecontext of histidine refers to chemical purity of histidine asunderstood in the art, e.g., as described in The Merck Index, 13^(th)ed., O'Neil et al. ed. (Merck & Co., 2001).

The pH of the formulation generally should not be equal to theisoelectric point of the particular antibody (including antibodyfragment thereof) to be used in the formulation (e.g., the isoelectricpoint of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 ranges from 8.65 to 8.89)and may range from about 5.0 to about 7.0, preferably about 5.5 to about6.5, more preferably about 5.8 to about 6.2, and most preferably about6.0.

In addition to histidine and an antibody (including antibody fragmentthereof) that immunospecifically binds to an IL-9 polypeptide, theformulations of the present invention may further comprise glycine at aconcentration of less than 150 mM, less than 100 mM, less than 75 mM,less than 50 mM, less than 25 mM, less than 10 mM, less than 5.0 mM, orless than 2.0 mM. In a specific embodiment, the formulations of thepresent invention further comprise glycine at a concentration of 1 mM to150 mM, 1 mM to 100 mM, 1 mM to 75 mM, 1 mM to 50 mM, 1 mM to 25 mM, 1mM to 10 mM, 1 mM to 5.0 mM, or 1 mM to 2.0 mM. The amount of glycine inthe formulation should not cause a significant buffering effect so thatantibody precipitation at its isoelectric point can be avoided. Glycinemay be also used in a form of pharmaceutically acceptable salt, such ashydrochloride, hydrobromide, sulfate, acetate, etc. The purity ofglycine should be at least 98%, preferably at least 99%, and mostpreferably 99.5%. As used herein, the term “purity” in the context ofglycine refers to chemical purity of glycine as understood in the art,e.g., as described in The Merck Index, 13^(th) ed., O'Neil et al. ed.(Merck & Co., 2001). In a specific embodiment, glycine is not includedin the formulations of the present invention.

Optionally, the formulations of the present invention may furthercomprise other excipients, such as saccharides (e.g., sucrose, mannose,trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). In oneembodiment, the other excipient is a saccharide. In a specificembodiment, the saccharide is sucrose, which is at a concentrationranging from between about 1% to about 20%, preferably about 5% to about15%, and more preferably about 8% to 10% of the formulation. In anotherembodiment, the saccharide is sucrose, which is at a concentration of1%, 3%, 5%, 8%, 10%, 15%, or 20% of the formulation. In anotherembodiment, an excipient is a polyol. Preferably, however, the liquidformulations of the present invention do not contain mannitol. In aspecific embodiment, the polyol is polysorbate (e.g., Tween 20), whichis at a concentration ranging from between about 0.001% to about 1%,preferably, about 0.01% to about 0.1% of the formulation. In a specificembodiment, the polyol is polysorbate (e.g., Tween 20), which is at aconcentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%,or 1% of the formulation.

The liquid formulations of the present invention exhibit stability atthe temperature range of 38° C.-42° C. for at least 15 days and, in someembodiments, not more than 25 days, at the temperature range of 20°C.-24° C. for at least 6 months, at the temperature range of 2° C.-8° C.(in particular, at 4° C.) for at least 6 months, at least 1 year, atleast 1.5 years, at least 2 years, at least 2.5 years, at least 3 yearsor at least 4 years, and at the temperature of −20° C. for at least 2years, at least 3 years, at least 4 years, or at least 5 years, asassessed by high performance size exclusion chromatography (HPSEC).Namely, the liquid formulations of the present invention have low toundetectable levels of aggregation and/or fragmentation, as definedherein, after the storage for the defined periods as set forth above.Preferably, no more than 5%, no more than 4%, no more than 3%, no morethan 2%, no more than 1%, and most preferably no more than 0.5% of theantibody (including antibody fragment thereof) forms an aggregate asmeasured by HPSEC, after the storage for the defined periods as setforth above. Furthermore, liquid formulations of the present inventionexhibit almost no loss in biological activities of the antibody(including antibody fragment thereof) during the prolonged storage underthe condition described above, as assessed by various immunologicalassays including, for example, enzyme-linked immunosorbent assay (ELISA)and radioimmunoassay to measure the ability of the antibody (includingantibody fragment thereof) to immunospecifically bind to an IL-9polypeptide. The liquid formulations of the present invention retainafter the storage for the above-defined periods more than 80%, more than85%, more than 90%, more than 95%, more than 98%, more than 99%, or morethan 99.5% of the initial biological activities (e.g., the ability tobind to an IL-9 polypeptide) of the formulation prior to the storage. Insome embodiments, the liquid formulations of the present inventionretain after the storage for the above-defined periods at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, or atleast 99.5% of the biological activity (e.g., the ability to bind to anIL-9 polypeptide) compared to a reference antibody representing theantibody prior to the storage.

The liquid formulations of the present invention can be prepared as unitdosage forms. For example, a unit dosage per vial may contain 1 ml, 2ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml ofdifferent concentrations of an antibody or antibody fragment thatimmunospecifically binds to an IL-9 polypeptide ranging from about 15mg/ml to about 300 mg/ml, about 50 mg/ml to about 300 mg/ml, about 75mg/ml to about 300 mg/ml, about 95 mg/ml to about 300 mg/ml, about 100mg/ml to about 300 mg/ml, about 150 mg/ml to about 300 mg/ml, about 200mg/ml to about 300 mg/ml, about 100 mg/ml to about 200 mg/ml, about 100mg/ml to about 150 mg/ml, or about 100 mg/ml to about 175 mg/ml. Ifnecessary, these preparations can be adjusted to a desired concentrationby adding a sterile diluent to each vial.

The invention encompasses stable liquid formulations comprising a singleantibody (including antibody fragment thereof) that immunospecificallybinds to an IL-9 polypeptide. The invention also encompasses stableliquid formulations comprising two or more antibodies (includingantibody fragments thereof) that immunospecifically bind to an IL-9polypeptide. In a specific embodiment, a stable liquid formulation ofthe invention comprises 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or a fragmentthereof that immunospecifically binds to an IL-9 polypeptide. In anotherembodiment, a stable liquid formulation of the invention comprises twoor more antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide, wherein one of theantibodies (including antibody fragments thereof) is 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or an antigen-binding fragment thereof. In an alternativeembodiment, a stable liquid formulation of the invention comprises twoor more antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide, with the proviso thatthe antibodies (including antibody fragments thereof) do not include4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or an antigen-binding fragmentthereof.

5.1.1. IL-9 Antibodies

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide (preferably, a human IL-9polypeptide). In particular, the invention provides for the formulationsof the following antibodies that immunospecifically bind to an IL-9polypeptide: 4D4 or an antigen-binding fragment thereof, 4D4 H2-1 D11 oran antigen-binding fragment thereof, 4D4com-XF-9 or an antigen-bindingfragment thereof, 4D4com-2F9 or an antigen-binding fragment thereof, 7F3or an antigen-binding fragment thereof, 71A10 or an antigen-bindingfragment thereof, 7F3 22D3 or an antigen-binding fragment thereof,7F3com-2H2 or an antigen-binding fragment thereof, 7F3com-3H5 or anantigen-binding fragment thereof, and 7F3com-3D4 or an antigen-bindingfragment thereof. In a preferred embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide is 7F3com-2H2 or anantigen-binding fragment thereof (e.g., one or more CDRs of 7F3com-2H2).The constant regions for 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9,71A10, 7F3 22D3, 7F3com, 7F3com-2H2, 7F3com-3H5, and 7F3com-3D4 areidentical to the constant regions of palizvizumab (MedImmune, Inc.) IgG₁(see U.S. Pat. No. 5,824,307, issued Oct. 20, 1998).

The present invention provides formulations of antibodies thatimmunospecifically bind an IL-9 polypeptide, said antibodies comprisinga VH domain having an amino acid sequence of the VH domain of 4D4 (FIG.1A; SEQ ID NO.: 7), 4D4 H2-1 D11 (FIG. 2A; SEQ ID NO.: 9), 4D4com-XF-9(FIG. 3A; SEQ ID NO.: 15), 4D4com-2F9 (FIG. 4A; SEQ ID NO.: 17), 7F3(FIG. 5A; SEQ ID NO.: 21), 71A10 (FIG. 6A; SEQ ID NO.: 23), 7F3 22D3(FIG. 7A; SEQ ID NO.: 21), 7F3com-2H2 (FIG. 8A; SEQ ID NO.: 27),7F3com-3H5 (FIG. 10A; SEQ ID NO.: 29), or 7F3com-3D4 (FIG. 11A; SEQ IDNO.: 31). In a preferred embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide comprises a VH domain having an amino acidsequence of the VH domain of 7F3com-2H2 (FIG. 8A; SEQ ID NO: 27).

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a VH CDR having an amino acid sequence of any one of the VHCDRs listed in Table 1, supra. In particular, the invention providesantibodies that immunospecifically bind to an IL-9 polypeptide, saidantibodies comprising (or alternatively, consisting of) one, two, three,four, five or more VH CDRs having an amino acid sequence of any of theVH CDRs listed in Table 1, infra. In one embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VH CDR1having the amino acid sequence of SEQ ID NO.: 1, SEQ ID NO.: 11, SEQ IDNO.: 19, or SEQ ID NO.: 26. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VH CDR2having the amino acid sequence of SEQ ID NO.: 2 or SEQ ID NO.: 10. Inanother embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR3 having the amino acid sequence of SEQ IDNO.: 3 or SEQ ID NO.: 12. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VH CDR1having the amino acid sequence of SEQ ID NO.: 1, SEQ ID NO.: 11, SEQ IDNO.: 19, or SEQ ID NO.: 26 and a VH CDR2 having the amino acid sequenceof SEQ ID NO.: 2 or SEQ ID NO.: 10. In another embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide comprises a VH CDR1having the amino acid sequence of SEQ ID NO.: 1, SEQ ID NO.: 1, SEQ IDNO.: 19, or SEQ ID NO.: 26 and a VH CDR3 having the amino acid sequenceof SEQ ID NO.: 3 or SEQ ID NO.: 12. In another embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide comprises a VH CDR2having the amino acid sequence of SEQ ID NO.: 2 or SEQ ID NO.: 10 and aVH CDR3 having the amino acid sequence of SEQ ID NO.: 3 or SEQ ID NO.:12. In another embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide comprises a VH CDR1 having the amino acid sequenceof SEQ ID NO.: 1, SEQ ID NO.: 11, SEQ ID NO.: 19, or SEQ ID NO.: 26, aVH CDR2 having the amino acid sequence of SEQ ID NO.: 2 or SEQ ID NO.:10, and a VH CDR3 having the amino acid sequence of SEQ ID NO.: 3 or SEQID NO.: 12.

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a VL domain having an amino acid sequence of the VL domainfor 4D4 (FIG. 1B; SEQ ID NO.: 8), 4D4 H2-1 D11 (FIG. 2B; SEQ ID NO.: 8),4D4com-XF-9 (FIG. 3B; SEQ ID NO.: 16), 4D4com-2F9 (FIG. 4B; SEQ ID NO.:18), 7F3 (FIG. 5B; SEQ ID NO.: 22), 71A10 (FIG. 6B; SEQ ID NO.: 24), 7F322D3 (FIG. 7B; SEQ ID NO.: 25), 7F3com-2H2 (FIG. 8B; SEQ ID NO.: 28),7F3com-3H5 (FIG. 10B; SEQ ID NO.: 30), or 7F3com-3D4 (FIG. 11B; SEQ IDNO.: 32). In a preferred embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide comprises a VL domain having an amino acidsequence of the VL domain for 7F3com-2H2 (FIG. 8B; SEQ ID NO.: 28).

The present invention also provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a VL CDR having an amino acid sequence of any one of the VLCDRs listed in Table 1, supra. In particular, the invention providesantibodies that immunospecifically bind to an IL-9 polypeptide, saidantibodies comprising (or alternatively, consisting of) one, two, threeor more VL CDRs having an amino acid sequence of any of the VL CDRslisted in Table 1, infra. In one embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VL CDR1having the amino acid sequence of SEQ ID NO.: 4 or SEQ ID NO.: 13. Inanother embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VL CDR2 having the amino acid sequence of SEQ IDNO.: 5 or SEQ ID NO.: 14. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VL CDR3having the amino acid sequence of SEQ ID NO.: 6 or SEQ ID NO.: 20. Inanother embodiment, an antibody of that immunospecifically binds to anIL-9 polypeptide comprises a VL CDR1 having the amino acid sequence ofSEQ ID NO.: 4 or SEQ ID NO.: 13 and a VL CDR2 having the amino acidsequence of SEQ ID NO.: 5 or SEQ ID NO.: 14. In another embodiment of anantibody that immunospecifically binds to an IL-9 polypeptide comprisesa VL CDR1 having the amino acid sequence of SEQ ID NO.: 4 or SEQ ID NO.:13 and a VL CDR3 having the amino acid sequence of SEQ ID NO.: 6 or SEQID NO.: 20. In another embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide comprises a VL CDR2 having the amino acidsequence of SEQ ID NO.: 5 or SEQ ID NO.: 14 and a VL CDR3 having theamino acid sequence of SEQ ID NO.: 6 or SEQ ID NO.: 20. In anotherembodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VL CDR1 having the amino acid sequence of SEQ IDNO.: 4 or SEQ ID NO.: 13, a VL CDR2 having the amino acid sequence ofSEQ ID NO.: 5 or SEQ ID NO.: 14, and a VL CDR3 having the amino acidsequence of SEQ ID NO.: 6 or SEQ ID NO.:20, being a part of theantibody.

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a VH domain disclosed herein combined with a VL domaindisclosed herein, or other VL domain (e.g., a VL domain disclosed inU.S. provisional application Ser. No. 60/371,683, filed Apr. 12, 2002and U.S. provisional application Ser. No. 60/371,728, filed Apr. 12,2002, each of which is incorporated herein by reference in itsentirety). The present invention also provides antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a VL domain disclosed herein combined with a VH domaindisclosed herein, or other VH domain (e.g., a VH domain disclosed inU.S. provisional application Ser. No. 60/371,683, filed Apr. 12, 2002and U.S. provisional application Ser. No. 60/371,728, filed Apr. 12,2002, both of which are incorporated by reference herein in theirentireties).

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising (or alternatively, consisting of) a VH CDR listed in Table 1,supra and a VL CDR disclosed in U.S. provisional application Ser. No.60/371,683, filed Apr. 12, 2002 and U.S. provisional application Ser.No. 60/371,728, filed Apr. 12, 2002. The present invention also providesantibodies that immunospecifically bind to an IL-9 polypeptide, saidantibodies comprising (or alternatively, consisting of) a VL CDR listedin Table 1, supra and a VH CDR disclosed in U.S. provisional applicationSer. No. 60/371,683, filed Apr. 12, 2002 and U.S. provisionalapplication Ser. No. 60/371,728, filed Apr. 12, 2002. The inventionfurther provides antibodies that immunospecifically bind to an IL-9polypeptide, said antibodies comprising combinations of VH CDRs and VLCDRs described herein and disclosed in U.S. provisional application Ser.No. 60/371,683, filed Apr. 12, 2002 and U.S. provisional applicationSer. No. 60/371,728, filed Apr. 12, 2002.

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising one or more VH CDRs and one or more VL CDRs listed in Table1, supra. In particular, the invention provides an antibody thatimmunospecifically binds to an IL-9 polypeptide, said antibodycomprising (or alternatively, consisting of) a VH CDR1 and a VL CDR1; aVH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and a VLCCDR1; V DR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VHCDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VHCDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VHCDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, a VHCDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1, a VLCDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, a VLCDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and a VLCDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VHCDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combination thereof ofthe VH CDRs and VL CDRs listed in Table 1, supra.

In one embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR1 having the amino acid sequence of SEQ IDNO.: 1, SEQ ID NO.: 1, SEQ ID NO.: 19, or SEQ ID NO.: 26 and a VL CDR1having the amino acid sequence of SEQ ID NO.: 4 or SEQ ID NO.: 13. Inanother embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR1 having the amino acid sequence of SEQ IDNO.: 1, SEQ ID NO.: 1, SEQ ID NO.: 19, or SEQ ID NO.: 26 and a VL CDR2having the amino acid sequence of SEQ ID NO.: 5 or SEQ ID NO.: 14. Inanother embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR1 having the amino acid sequence of SEQ IDNO.: 1, SEQ ID NO.: 1, SEQ ID NO.: 19, or SEQ ID NO.: 26 and a VL CDR3having an amino acid sequence of SEQ ID NO.: 6 or SEQ ID NO.: 20.

In one embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR2 having the amino acid sequence of SEQ IDNO.: 2 or SEQ ID NO.: 10 and a VL CDR1 having the amino acid sequence ofSEQ ID NO.: 4 or SEQ ID NO.: 13. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VH CDR2having the amino acid sequence of SEQ ID NO.: 2 or SEQ ID NO.: 10 and aVL CDR2 having the amino acid sequence of SEQ ID NO.: 5 or SEQ ID NO.:14. In another embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide comprises a VH CDR2 having the amino acid sequenceof SEQ ID NO.: 2 or SEQ ID NO.: 10 and a VL CDR3 having an amino acidsequence of SEQ ID NO.: 6 or SEQ ID NO.: 20.

In one embodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises a VH CDR3 having the amino acid sequence of SEQ IDNO.: 3 or SEQ ID NO.: 12 and a VL CDR1 having the amino acid sequence ofSEQ ID NO.: 4 or SEQ ID NO.: 13. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises a VH CDR3having the amino acid sequence of SEQ ID NO.: 3 or SEQ ID NO.: 12 and aVL CDR2 having the amino acid sequence of SEQ ID NO.: 5 or SEQ ID NO.:14. In another embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide comprises a VH CDR3 having the amino acid sequenceof SEQ ID NO.: 3 or SEQ ID NO.: 12 and a VL CDR3 having an amino acidsequence of SEQ ID NO.: 6 or SEQ ID NO.: 20.

The present invention provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising derivatives of the VH domains, VH CDRs, VL domains, or VLCDRs described herein that immunospecifically bind to an IL-9polypeptide. Standard techniques known to those of skill in the art canbe used to introduce mutations (e.g., deletions, additions, and/orsubstitutions) in the nucleotide sequence encoding an antibody of theinvention, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis which results in amino acid substitutions.Preferably, the derivatives include less than 25 amino acidsubstitutions, less than 20 amino acid substitutions, less than 15 aminoacid substitutions, less than 10 amino acid substitutions, less than 5amino acid substitutions, less than 4 amino acid substitutions, lessthan 3 amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original molecule. In a preferred embodiment, thederivatives have conservative amino acid substitutions are made at oneor more predicted non-essential amino acid residues (i.e., amino acidresidues which are not critical for the antibody to immunospecificallybind to an IL-9 polypeptide). A “conservative amino acid substitution”is one in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded antibody can be expressed and the activity ofthe antibody can be determined.

The present invention provides for formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising the amino acid sequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4with one or more amino acid residue substitutions in the variable light(VL) domain and/or variable heavy (VH) domain. The present inventionalso provides for antibodies that immunospecifically bind to an IL-9polypeptide, said antibodies comprising the amino acid sequence of 4D4,4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4 with one or more amino acid residuesubstitutions in one or more VL CDRs and/or one or more VH CDRs. Thepresent invention also provides for antibodies that immunospecificallybind to an IL-9 polypeptide, said antibodies comprising the amino acidsequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4, or a VH and/or VL domainthereof with one or more amino acid residue substitutions in one or moreVH frameworks and/or one or more VL frameworks. The antibody generatedby introducing substitutions in the VH domain, VH CDRs, VL domain VLCDRs and/or frameworks of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9,7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 can betested in vitro and/or in vivo, for example, for its ability to bind toan IL-9 polypeptide, or for its ability to inhibit or reduce IL-9mediated cell proliferation, or for its ability to prevent, treat and/orameliorate an autoimmune disorder, an inflammatory disorder, aproliferative disorder or a respiratory infection, or a symptom thereof.

In a specific embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide comprises a nucleotide sequence that hybridizes tothe nucleotide sequence encoding 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4,or an antigen-binding fragment thereof under stringent conditions, e.g.,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., under highly stringent conditions, e.g.,hybridization to filter-bound nucleic acid in 6×SSC at about 45° C.followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., orunder other stringent hybridization conditions which are known to thoseof skill in the art (see, for example, Ausubel, F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. 1, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3).

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide comprises an amino acid sequence of a VH domain or anamino acid sequence a VL domain encoded by a nucleotide sequence thathybridizes to the nucleotide sequence encoding the VH or VL domains of4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 under stringent conditionsdescribed herein or under other stringent hybridization conditions whichare known to those of skill in the art. In another embodiment, anantibody that immunospecifically binds to an IL-9 polypeptide comprisesan amino acid sequence of a VH domain and an amino acid sequence of a VLdomain encoded by a nucleotide sequence that hybridizes to thenucleotide sequence encoding the VH and VL domains of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4 under stringent conditions described herein or under otherstringent hybridization conditions which are known to those of skill inthe art. In another embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide comprises an amino acid sequence of a VHCDR or an amino acid sequence of a VL CDR encoded by a nucleotidesequence that hybridizes to the nucleotide sequence encoding any one ofthe VH CDRs or VL CDRs listed in Table 1, supra under stringentconditions described herein or under other stringent hybridizationconditions which are known to those of skill in the art. In anotherembodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises an amino acid sequence of a VH CDR and an aminoacid sequence of a VL CDR encoded by nucleotide sequences that hybridizeto the nucleotide sequences encoding any one of the VH CDRs listed inTable 1, supra, and any one of the VL CDRs listed Table 1, supra, understringent conditions described herein or under other stringenthybridization conditions which are known to those of skill in the art.

In a specific embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide comprises an amino acid sequence that is at least35%, preferably at least 40%, at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to theamino acid sequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4, or anantigen-binding fragment thereof. In another embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide comprises an aminoacid sequence of a VH domain that is at least 35%, preferably at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% identical to the VH domain of 4D4,4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5 or 7F3com-3D4. In another embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide comprises an amino acidsequence of a VL domain that is at least 35%, preferably at least 40%,at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to the VL domain of 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5 or 7F3com-3D4.

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide comprises an amino acid sequence of one or more VL CDRsthat are at least 35%, preferably at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least99% identical to any of the VL CDRs listed in Table 1, supra. In anotherembodiment, an antibody that immunospecifically binds to an IL-9polypeptide comprises an amino acid sequence of one or more VL CDRs thatare at least 35%, preferably at least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to any of one of the VL CDRs listed in Table 1, supra.

The present invention encompasses formulations of antibodies thatcompete with an antibody described herein for binding to an IL-9polypeptide. In particular, the present invention encompasses antibodiesthat compete with 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 or anantigen-binding fragment thereof for binding to the IL-9 polypeptide. Ina specific embodiment, the invention encompasses an antibody thatreduces the binding of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 to an IL-9polypeptide by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or more, or 5 to 15%, 10 to 25%, 25% to 50%, 45 to 75%, or 75to 99% relative to a control such as PBS in the competition assaydescribed herein or competition assays well known in the art. In anotherembodiment, the invention encompasses formulations of an antibody thatreduces binding of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 to an IL-9polypeptide by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or more, or 5 to 15%, 10 to 25%, 25% to 50%, 45 to 75%, or 75to 99% relative to a control such as PBS in an ELISA competition assay.In a preferred embodiment, an ELISA competition assay may be performedin the following manner: recombinant IL-9 is prepared in PBS at aconcentration of 10 μg/ml. 100 μl of this solution is added to each wellof an ELISA 98-well microtiter plate and incubated overnight at 4-8° C.The ELISA plate is washed with PBS supplemented with 0.1% Tween toremove excess recombinant IL-9. Non-specific protein-proteininteractions are blocked by adding 100 μl of bovine serum albumin (BSA)prepared in PBS to a final concentration of 1%. After one hour at roomtemperature, the ELISA plate is washed. Unlabeled competing antibodiesare prepared in blocking solution at concentrations ranging from 1 μg/mlto 0.01 μg/ml. Control wells contain either blocking solution only orcontrol antibodies at concentrations ranging from 1 μg/ml to 0.01 μg/ml.Test antibody (e.g., 7F3com-2H2) labeled with horseradish peroxidase isadded to competing antibody dilutions at a fixed final concentration of1 μg/ml. 100 μl of test and competing antibody mixtures are added to theELISA wells in triplicate and the plate is incubated for 1 hour at roomtemperature. Residual unbound antibody is washed away. Bound testantibody is detected by adding 100 μl of horseradish peroxidasesubstrate to each well. The plate is incubated for 30 min. at roomtemperature, and absorbance is read using an automated plate reader. Theaverage of triplicate wells is calculated. Antibodies which compete wellwith the test antibody reduce the measured absorbance compared withcontrol wells. In another preferred embodiment, the inventionencompasses an antibody that reduces the binding of 7F3com-2H2 to anIL-9 polypeptide by at least 5%, preferably at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or more, or 5 to 15%, 10 to 25%, 25% to 50%, 45 to 75%, or 75to 99% relative to a control such as PBS in an ELISA competition assay(described above).

In another embodiment, the invention encompasses formulations of anantibody that reduces the binding of an antibody comprising(alternatively, consisting of) an antigen-binding fragment (e.g., a VHdomain, a VH CDR, a VL domain or a VL CDR) of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4 to an IL-9 polypeptide by at least 2%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or more, or 5 to 15%, 10 to 25%, 25% to50%, 45 to 75%, or 75 to 99% relative to a control such as PBS in acompetition assay described herein or well-known to one of skill in theart. In another embodiment, the invention encompasses an antibody thatreduces the binding of an antibody comprising (alternatively, consistingof) an antigen-binding fragment (e.g., a VH domain, VL domain, a VH CDR,or a VL CDR) of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10,7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 to an IL-9 polypeptide byat least 2%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% ormore, or 5 to 15%, 10 to 25%, 25% to 50%, 45 to 75%, or 75 to 99%relative to a control such as PBS in an ELISA competition assay. In apreferred embodiment, the invention encompasses an antibody that reducesthe binding of an antibody comprising (alternatively, consisting of) anantigen-binding fragment of 7F3com-2H2 to an IL-9 polypeptide by atleast 2%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% ormore, or 5 to 15%, 10 to 25%, 25% to 50%, 45 to 75%, or 75 to 99%relative to a control such as PBS in an ELISA competition assay.

The present invention encompasses formulations of polypeptides orproteins comprising (alternatively, consisting of) VH domains thatcompete with the VH domain of 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9,7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 for bindingto an IL-9 polypeptide. The present invention also encompassesformulations of polypeptides or proteins comprising (alternatively,consisting of) VL domains that compete with a VL domain of 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5 or 7F3com-3D4 for binding to an IL-9 polypeptide.

The present invention encompasses formulations of polypeptides orproteins comprising (alternatively, consisting of) VH CDRs that competewith a VH CDR listed in Table 1, supra, for binding to an IL-9polypeptide. The present invention also encompasses polypeptides orproteins comprising (alternatively consisting of) VL CDRs that competewith a VL CDR listed in Table 1, supra for binding to an IL-9polypeptide.

The antibodies that immunospecifically bind to an IL-9 polypeptideinclude derivatives that are modified, i.e., by the covalent attachmentof any type of molecule to the antibody such that covalent attachment.For example, but not by way of limitation, the antibody derivativesinclude antibodies that have been modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to, specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

The present invention also provides formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising a framework region known to those of skill in the art (e.g.,a human or non-human framework). The framework regions may be naturallyoccurring or consensus framework regions. Preferably, the fragmentregion of an antibody of the invention is human (see, e.g., Chothia etal., 1998, J. Mol. Biol. 278:457-479 for a listing of human frameworkregions, which is incorporated herein by reference in its entirety).

The present invention encompasses formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising the amino acid sequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4with mutations (e.g., one or more amino acid substitutions) in theframework regions. In certain embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide comprise the amino acidsequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4 with one or more amino acidresidue substitutions in the framework regions of the VH and/or VLdomains. Preferably, the amino acid substitutions in the frameworkregion improve binding of the antibody to an IL-9 polypeptide.

In a specific embodiment, formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide comprise the amino acidsequence of one or more of the CDRs of 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4,a VH framework region 1 having the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKAS (SEQ ID NO.: 33) or QVQLVQSGAEVKKPGSSVKVSCKAS(SEQ ID NO.: 37), a VH framework region 2 having the amino acid sequenceof WVRQAPGQGLEWMG (SEQ ID NO.: 34), a VH framework region 3 regionhaving the amino acid sequence of RVTMTRDTSTSTVYM ELSSLRSEDTAVYYCAR (SEQID.: 35) or RVTITADESTSTAYMELSSLRSED TAVYYCAR (SEQ ID NO.: 38), and a VHframework region 4 having the amino acid sequence of WGQGTLVTVSS (SEQ IDNO.: 36). In another embodiment, antibodies that immunospecifically bindto an IL-9 polypeptide comprise the amino acid sequence of one or moreof the CDRs of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10,7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4, a VL framework region 1having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO.:39), a VL framework region 2 having the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO.: 40), a VL framework region 3 region havingthe amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO.:41), and a VL framework region 4 region having the amino acid sequenceof FGGGTKVEIK (SEQ ID NO.: 42). In yet another embodiment, antibodiesthat immunospecifically bind to an IL-9 polypeptide comprise the aminoacid sequence of one or more of the CDRs of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4, a VH framework region 1 having the amino acid sequence ofSEQ ID NO.: 33 or SEQ ID NO.: 37, a VH framework region 2 having theamino acid sequence of SEQ ID NO.: 34, a VH framework region 3 havingthe amino acid sequence of SEQ ID NO.: 35 or 38, a VH framework region 4having the amino acid sequence of SEQ ID NO.: 36, a VL framework region1 having the amino acid sequence of SEQ ID NO.: 39, a VL frameworkregion 2 having the amino acid sequence of SEQ ID NO.: 40, a VLframework region 3 having the amino acid sequence of SEQ ID NO.: 41, anda VL framework region 4 having the amino acid sequence of SEQ ID NO.:42.

The present invention also encompasses formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide, said antibodiescomprising the amino acid sequence of 4D4, 4D4 H2-1 D11, 4D4com-XF-9,4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4with mutations (e.g., one or more amino acid residue substitutions) inthe variable and framework regions. Preferably, the amino acidsubstitutions in the variable and framework regions improve binding ofthe antibody to an IL-9 polypeptide.

The present invention also provides formulations of antibodies of theinvention that comprise constant regions known to those of skill in theart. Preferably, the constant regions of an antibody of the invention orfragment thereof are human.

The invention encompasses formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide expressed by an immunecell such as an activated T cell or a mast cell. The invention alsoencompasses antibodies that immunospecifically bind to an IL-9polypeptide and modulate an activity or function of T cells, B cells,mast cells, neutrophils, and/or eosinophils. The invention furtherencompasses antibodies that immunospecifically bind to an IL-9polypeptide and inhibit or reduce the infiltration of inflammatory cellsinto a tissue, joint, or organ of a subject and/or inhibit or reduceepithelial cell hyperplasia.

The invention encompasses formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide found in the milieu,i.e., not bound to an IL-9R or a subunit thereof. The invention alsoencompasses antibodies that immunospecifically bind to an IL-9polypeptide bound to a soluble IL-9Ra subunit. The invention furtherencompasses antibodies that immunospecifically bind to an IL-9polypeptide bound to a cellular membrane-bound IL-9R or a subunitthereof.

In one embodiment, an antibody that immunospecifically binds to an IL-9polypeptide inhibits and/or reduces the interaction between the IL-9polypeptide and the IL-9R or a subunit thereof by approximately 25%,preferably approximately 30%, approximately 35%, approximately 45%,approximately 50%, approximately 55%, approximately 60%, approximately65%, approximately 70%, approximately 75%, approximately 80%,approximately 85%, approximately 90%, approximately 95%, orapproximately 98% relative to a control such as PBS or an IgG controlantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art (e.g., an immunoassay such as anELISA). In an alternative embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide does not inhibit theinteraction between an IL-9 polypeptide and the IL-9R or a subunitthereof relative to a control such as PBS or an IgG control antibody inan in vivo and/or in vitro assay described herein or well-known to oneof skill in the art (e.g., an immunoassay such as an ELISA). In anotherembodiment, an antibody that immunospecifically binds to an IL-9polypeptide inhibits the interaction between the IL-9 polypeptide andthe IL-9R by less than 20%, less than 15%, less than 10%, or less than5% relative to a control such as PBS or an IgG control antibody using,for example, an immunoassay such as an ELISA.

In one embodiment, antibodies that immunospecifically bind to an IL-9polypeptide inhibit or reduce the interaction between the IL-9polypeptide and the IL-9R or one or more subunits thereof by at least25%, preferably, at least 30%, at least 35%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as phosphate buffered saline (“PBS”) or anIgG control antibody in an in vivo and/or in vitro assay describedherein or well-known to one of skill in the art (e.g., a cellproliferation assay using an IL-9 dependent cell line such as an IL-9dependent mouse T cell line expressing the human IL-9R). In analternative embodiment, antibodies that immunospecifically bind to anIL-9 polypeptide do not inhibit the interaction between an IL-9polypeptide and the IL-9R or one or more subunits thereof relative to acontrol such as PBS or an IgG control antibody in an in vivo and/or invitro assay described herein or well-known to one of skill in the art(e.g., a cell proliferation assay using an IL-9 dependent cell line suchas an IL-9 dependent mouse T cell line expressing the human IL-9R). Inanother embodiment, antibodies that immunospecifically bind to an IL-9polypeptide inhibit the interaction between the IL-9 polypeptide and theIL-9R or one or more subunits thereof by less than 20%, less than 15%,less than 10%, or less than 5% relative to a control such as PBS or anIgG control antibody in vivo and/or in vitro assay described herein orwell-known to one of skill in the art, (e.g., a cell proliferation assayusing an IL-9 dependent cell line such as an IL-9 dependent mouse T cellline expressing the human IL-9R).

The present invention encompasses formulations of antibodies thatimmunospecifically bind to an IL-9 polypeptide and do not induce orreduce cytokine expression and/or release relative to a control such asPBS or an IgG control antibody in an in vivo and/or in vitro assaydescribed herein or well-known to one of skill in the art. In oneembodiment, antibodies that immunospecifically bind to an IL-9polypeptide and do not induce an increase in the concentration cytokinessuch as, e.g., IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15,and IL-23 in the serum of a subject administered such an antibodyrelative to the concentration of such cytokines in the serum of asubject administered a ctrol such as PBS or an IgG control antibody. Inan alternative embodiment, antibodies that immunospecifically bind to anIL-9 polypeptide induce cytokine expression and/or release relative to acontrol such as PBS or an IgG control antibody in an in vitro and/or invivo assay described herein or well-known to one of skill in the art. Ina specific embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide induces an increase in the concentration of cytokinessuch as, e.g., IFN-γ, IL-2, IL-12, and IL-15 in the serum of a subjectadministered such an antibody relative to the concentration of suchcytokines in the serum of a subject administered a control such as PBSor an IgG control antibody. In another specific embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide induces an increasein the concentration of cytokines produced by Th1 cells, such as IFN-γand IL-12, in a subject administered such an antibody relative to theconcentration of such cytokines in the serum of a subject administered acontrol such as PBS or an IgG control antibody. In another specificembodiment, an antibody that immunospecifically binds to an IL-9polypeptide induces a decrease in the concentration of cytokines suchas, e.g., IL-4, IL-5, IL-10, IL-13, and IL-23 in the serum of a subjectadministered such an antibody relative to the concentration of suchcytokines in the serum of a subject administered a control such as PBSor an IgG control antibody. In another specific embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide induces a decreasein the concentration of cytokines produced by mast cells, such as TNF-α,IL-4, and IL-13, in the serum of a subject administered such an antibodyrelative to the concentration of such cytokines in the serum of asubject administered a control such as PBS or an IgG control antibody.In yet another specific embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide induces a decrease in the concentration ofcytokines produced by Th2 cells, such as IL-4, IL-5, IL-13, and IL-10,in the serum of a subject administered such an antibody relative to theconcentration of such cytokines in the serum of a subject administered acontrol such as PBS or an IgG control antibody. Serum concentrations ofa cytokine can be measured by any technique well-known to one of skillin the art such as, e.g., ELISA or Western blot assay.

In one embodiment, antibodies that immunospecifically bind to an IL-9polypeptide reduce and/or inhibit proliferation of inflammatory cells(e.g., mast cells, T cells, B cells, macrophages, neutrophils,basophils, and/or eosinophils) by at least 25%, preferably at least 30%,at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% relative to a control such asPBS or an IgG control antibody in an in vivo and/or in vitro assaydescribed herein or well-known to one of skill in the art (e.g., atrypan blue assay or ³H-thymidine assay). In another embodiment,antibodies that immunospecifically bind to an IL-9 polypeptide reduceand/or inhibit infiltration of inflammatory cells into the upper and/orlower respiratory tracts by at least at least 25%, preferably at least30%, at least 35%, at least 40%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 98% relative to a controlsuch as PBS or an IgG control antibody in an in vivo and/or in vitroassay described herein or well-known to one of skill in the art. In yetanother embodiment, antibodies that immunospecifically bind to an IL-9polypeptide reduce and/or inhibit infiltration of inflammatory cellsinto the upper and/or respiratory tracts by at least 25%, preferably atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or an IgG control antibody in an in vivo and/or invitro assay described herein or well known in the art and reduce and/orinhibit proliferation of inflammatory cells by at least by at least 25%,preferably at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or an IgG control antibody in an invivo and/or in vitro assay described herein or well-known to one ofskill in the art (e.g., a trypan blue assay or ³H-thymidine assay).

In certain embodiments, antibodies that immunospecifically bind to anIL-9 polypeptide reduce mast cell degranulation by at least 25%,preferably at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or an IgG control antibody in an invivo and/or in vitro assay described herein or well-known to one ofskill in the art (see, e.g., Windmiller and Backer, 2003, J. Biol. Chem.278:11874-78 for examples of mast cell degranulation assays). In otherembodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce mast cell activation by at least 25%,preferably at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or an IgG control antibody in an invivo and/or in vitro assay described herein or well-known to one ofskill in the art. In other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibit and/or reduce theexpression and/or release of products of mast cell activation and/ordegranulation by at least 25%, preferably at least 30%, at least 35%, atleast 40%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% relative to a control such as PBS or an IgGcontrol antibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art.

In a specific embodiment, antibodies that immunospecifically bind to anIL-9 polypeptide inhibit and/or reduce the expression, activity, serumconcentration, and/or release of mast cell proteases, such as chymaseand tryptase, by at least 25%, preferably at least 30%, at least 35%, atleast 40%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% relative to a control such as PBS or acontrol IgG antibody in an in vivo and/or in vitro assay describedherein or well known to one of skill in the art. In a preferredembodiment, mast cell activity may be measured by culturing primary mastcells or a mast cell line in vitro in the presence of 10 ng/ml of IL-9.Baseline levels of protease (e.g., chymase and tryptase) and leukotrieneare determined in the supernatant by commercially available ELISA kits.The ability of antibodies to modulate protease or leukotriene levels isassessed by adding an IL-9-reactive antibody or control antibodydirectly to cell cultures at a concentration of 1 μg/ml. Protease andleukotriene levels are assessed at 24 and 36 hour timepoints. In anotherspecific embodiment, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce the expression, activity, serumconcentration, and/or release of mast cell leukotrienes, such as C4, D4,and E4 by at least 25%, preferably at least 30%, at least 35%, at least40%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% relative to a control such as PBS or a control IgGantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art. In another specific embodiment,antibodies that immunospecifically bind to an IL-9 polypeptide inhibitand/or reduce the expression, activity, serum concentration, and/orrelease of mast cell cytokines, such as TNF-α, IL-4, and IL-13 by atleast 25%, preferably at least 30%, at least 35%, at least 40%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98% relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay described herein or well-known to one ofskill in the art (e.g., an ELISA or Western blot assay).

In other embodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce mast cell infiltration by at least25%, preferably at least 30%, at least 35%, at least 40%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay described herein or well-known in the art. Inother embodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce mast cell proliferation by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assay described herein or well-known to one of skill in the art(e.g., a trypan blue assay, FACS or ³H thymidine assay). In yet otherembodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce mast cell infiltration by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vitro and/orin vivo assay described herein or well-known in the art and inhibitand/or reduce mast cell proliferation at least 25%, at least 30%, atleast 35%, at least 40%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% relative to a control such asPBS or a control IgG antibody in an in vivo and/or in vitro assaydescribed herein or well-known to one of skill in the art (e.g., atrypan blue assay, FACS or ³H thymidine assay). In a preferredembodiment, reductions in mast cell infiltration may be measured in vivoby sensitizing animals to ovalbumin. Briefly, 100 μg of ovalbumincomplexed with aluminum adjuvant is administered subcutaneously on days1 and 21. Throughout the three-week sensitization procedure, animals areadministered an IL-9 reactive antibody or a control antibody at a 10mg/kg dose every 5 to 7 days. On days 29, 30 and 31, animals are exposedto ovalbumin without adjuvant by aerosol delivery, or alternatively, byintrasal instillation of 100 μl of a 1 μg/ml solution prepared in PBS.On day 31, 6 hours after the last ovalbumin challenge, animals areeuthanized and lung tissue is fixed by perfusion with formalin. Mastcell infiltration is assessed histologically by counting mast cells perfield in lung epithelial tissue sections. Using this experimentaldesign, mast cell precursors may be differentiated from mast cells inlung epithelium by assessing (for example) whether metachromaticgranules are present, and/or by immunohistochemistry usingdifferentiation-dependent cell surface markers (e.g., FcepsilonRI).

In other embodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce infiltration of mast cell precursorsin the upper and/or lower respiratory tracts by at least 25%, preferablyat least 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well-known in the art. In otherembodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce proliferation of mast cell precursorsby at least 25%, at least 30%, at least 35%, at least 40%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay described herein or well-known to one ofskill in the art (e.g., a trypan blue assay, FACS or ³H thymidineassay). In yet other embodiments, antibodies that immunospecificallybind to an IL-9 polypeptide inhibit and/or reduce infiltration of mastcell precursors into the upper and/or lower respiratory tracts by atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay described herein or well known in the art andinhibit and/or reduce proliferation of mast cell precursors at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assay described herein or well-known to one of skill in the art(e.g., a trypan blue assay, FACS or ³H thymidine assay). In a preferredembodiment, mast cell precursor infiltration may be measured in vivo bythe mast cell infiltration assay described supra.

In certain embodiments, antibodies that immunospecifically bind to anIL-9 polypeptide mediate depletion of peripheral blood T-cells byinducing an increase in apoptosis of T-cells, particularly Th2 cells. Ina preferred embodiment, Th2 T lymphocyte depletion may be measured invivo by sensitizing animals with ovalbumin. Briefly, 100 μg of ovalbumincomplexed with aluminum adjuvant is administered subcutaneously on days1 and 21. Throughout the three-week sensitization procedure, animals areadministered an IL-9 reactive antibody or a control antibody at a 10mg/kg dose every 5 to 7 days. On day 28, animals receive a 100 μg boostof ovalbumin protein without adjuvant intravenously. Two days followingthe intravenous boost, the animals are euthanized. Spleen cells arerecovered and analyzed by flow cytometry. Splenic Th2 T lymphocytes,identifiable by cytoplasmic staining for IL-4, should be reduced inanimals receiving an IL-9 neutralizing antibody compared with thecontrol antibody recipients. In another embodiment, antibodies thatimmunospecifically bind to an IL-9 polypeptide mediate inhibit and/orreduce Th1 and Th2 differentiation by at least 25%, preferably at least30%, at least 35%, at least 40%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 98% relative to a controlsuch as PBS or a control IgG antibody in an in vivo and/or in vitroassay described herein or well-known to one of skill in the art (e.g.,FACS). In certain embodiments, antibodies that immunospecifically bindto an IL-9 polypeptide inhibit and/or reduce T cell infiltration,particularly Th2 cell infiltration, in the upper and/or lowerrespiratory tracts by at least 25%, preferably at least 30%, at least35%, at least 40%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98% relative to a control such as PBS ora control IgG antibody in an in vivo and/or in vitro assay well-known toone of skill in the art. In other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibits and/or reduce Tcell proliferation by at least 25%, at least 30%, at least 35%, at least40%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% relative to a control such as PBS or a control IgGantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art (e.g., a trypan blue assay, FACSor ³H thymidine assay). In yet other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibit and/or reduce Tcell infiltration, particularly Th2 cell infiltration, in the upperand/or lower respiratory tracts by at least 25%, at least 30%, at least35%, at least 40%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98%, inhibit and/or reduce T cellproliferation, particularly Th2 cell proliferation, by at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98%, and/or increasesapoptosis of T cells relative to a control such as PBS or a control IgGantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art.

In certain embodiments, antibodies that immunospecifically bind to anIL-9 polypeptide reduce macrophage infiltration by at least 25%,preferably at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay well-known to one of skill in the art. In apreferred embodiment, reductions in macrophage infiltration may bemeasured in vivo by sensitizing animals to ovalbumin. Briefly, 100 μg ofovalbumin complexed with aluminum adjuvant is administeredsubcutaneously on days 1 and 21. Throughout the three-week sensitizationprocedure, animals are administered IL-9 reactive antibody or controlantibody at 10 mg/kg dose every 5 to 7 days. On days 29, 30 and 31,animals are exposed to ovalbumin without adjuvant by aerosol delivery,or alternatively, by intrasal instillation of 100 μl of a 1 μg/mlsolution prepared in PBS. On day 31, 6 hours after the last ovalbuminchallenge, animals are euthanized and lung tissue is fixed by perfusionwith formalin. Macrophage infiltration is assessed byimmunocytochemistry by counting CD14 positive cells per field in lungtissue sections. In other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibit and/or reducemacrophage proliferation by at least 25%, preferably, at least 30%, atleast 35%, at least 40%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% relative to a control such asPBS or a control IgG antibody in an in vivo and/or in vitro assaydescribed herein or well-known to one of skill in the art (e.g., atrypan blue assay, FACS or ³H thymidine assay). In yet otherembodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce macrophage infiltration by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assay described herein or well-known to one of skill in the artand inhibit and/or reduce macrophage proliferation at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well known to one of skill in the art.

In certain embodiments, antibodies that immunospecifically bind to anIL-9 polypeptide reduce B cell infiltration by at least 25%, preferablyat least 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well known to one of skill in the art.In a preferred embodiment, reductions in B lymphocyte infiltration maybe measured in vivo by systemically sensitizing animals to ovalbumin.Briefly, 100 μg of ovalbumin complexed with aluminum adjuvant isadministered subcutaneously on days 1 and 21. Throughout the three-weeksensitization procedure, animals are administered an IL-9 reactiveantibody or a control antibody at a 10 mg/kg dose every 5 to 7 days. Ondays 29, 30 and 31, animals are exposed to ovalbumin without adjuvant byaerosol delivery, or alternatively, by intrasal instillation of 100 μlof a 1 μg/ml solution prepared in PBS. On day 31, 6 hours after the lastovalbumin challenge, animals are euthanized and lung tissue is fixed byperfusion with formalin. B lymphocyte infiltration is assessed byimmunocytochemistry by counting CD19 positive cells per field in lungtissue sections. In other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibit and/or reduce Bcell proliferation by at least 25%, at least 30%, at least 35%, at least40%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% relative to a control such as PBS or a control IgGantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art (e.g., a trypan blue assay, FACSor ³H thymidine assay). In yet other embodiments, antibodies thatimmunospecifically bind to an IL-9 polypeptide inhibit and/or reduce Bcell infiltration by at least 25%, at least 30%, at least 35%, at least40%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% relative to a control such as PBS or a control IgGantibody in an in vivo and/or in vitro assay described herein orwell-known to one of skill in the art and inhibits and/or reduces B cellproliferation at least 25%, at least 30%, at least 35%, at least 40%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% relative to a control such as PBS or a control IgG antibody inan in vivo and/or in vitro assay described herein or well-known to oneof skill in the art.

In certain embodiments, antibodies that immunospecifically bind to anIL-9 polypeptide reduce eosinophil infiltration in the upper and/orlower respiratory tracts by at least 25%, preferably at least 30%, atleast 35%, at least 40%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% relative to a control such asPBS or a control IgG antibody in an in vivo and/or in vitro assaydescribed herein or well known to one of skill in the art (see, e.g., Liet al., 2000, Am. J. Respir. Cell Mol. Biol. 25:644-51). In otherembodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce eosinophil proliferation, by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assay described herein (see Section 5.6) or well known to oneof skill in the art (e.g., a trypan blue assay, FACS or ³H thymidineassay). In yet other embodiments, antibodies that immunospecificallybind to an IL-9 polypeptide inhibit and/or reduce eosinophilinfiltration by at least 25%, at least 30%, at least 35%, at least 40%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% relative to a control such as PBS or a control IgG antibody inan in vivo and/or in vitro assay described herein or well-known to oneof skill in the art and inhibits and/or reduces eosinophil proliferationat least 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%relative to a control such as PBS or a control IgG antibody in an invivo and/or in vitro assay described herein or well known to one ofskill in the art.

In other embodiments, antibodies that immunospecifically bind to an IL-9polypeptide reduce neutrophil infiltration by at least 25%, preferablyat least 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well known to one of skill in the art.In other embodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce neutrophil proliferation, by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assays described herein or well-known to one of skill in theart (e.g., a trypan blue assay, FACS or ³H thymidine assay). In yetother embodiments, antibodies that immunospecifically bind to an IL-9polypeptide inhibit and/or reduce neutrophil infiltration by at least25%, at least 30%, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 98% relativeto a control such as PBS or a control IgG antibody in an in vivo and/orin vitro assay described herein or well-known to one of skill in the artand inhibits and/or reduces neutrophil proliferation at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well-known to one of skill in the art.

In a preferred embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide neutralizes or inhibits IL-9 mediated biologicaleffects including, but not limited to inflammatory cell recruitment,epithelia hyperplasia, mucin production of epithelial cells, and mastcell activation, degranulation, proliferation, and/or infiltration.

In a specific embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide acts synergistically with a proteinaceous agent(e.g., a peptide, polypeptide, or protein (including an antibody))and/or a non-proteinaceous agent that antagonizes the expression,function, and/or activity of IgE to reduce or inhibit the activation,degranulation, proliferation, and/or infiltration of mast cells by atleast 25%, preferably, at least 30%, at least 35%, at least 40%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% relative to a control such as PBS or a control IgG antibody inan in vivo and/or in vitro assays described herein or well known to oneof skill in the art.

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide acts synergistically with a proteinaceous agent (e.g.,a peptide, polypeptide, protein (including an antibody)) and/or anon-proteinaceous agent that antagonizes the expression, function,and/or activity of a mast cell protease to reduce or inhibit theactivation, degranulation, proliferation, and/or infiltration of mastcells by at least 25%, preferably at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98% relative to a control such as PBS ora control IgG antibody in an in vivo and/or in vitro assay describedherein or well-known to one of skill in the art.

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide acts synergistically with a proteinaceous agent (e.g.,a peptide, polypeptide, and protein (including an antibody)) or anon-proteinaceous agent that antagonizes the expression, function,and/or activity of a stem cell factor to reduce or inhibit to reduce orinhibit the activation, degranulation, proliferation, and/orinfiltration of mast cells by at least 25%, preferably at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% relative to acontrol such as PBS or a control IgG antibody in an in vivo and/or invitro assay described herein or well-known to one of skill in the art.In a preferred embodiment, primary mast cells or a mast cell line iscultured in vitro in the presence of 1 ng/ml IL-9 plus 1 ng/ml stem cellfactor. Baseline levels of protease (e.g., chymase and tryptase) andleukotriene are determined in the supernatant by commercially availableELISA kits. The ability of antibodies to modulate protease orleukotriene levels is assessed by adding IL-9 reactive antibody orcontrol antibody directly to cell cultures at a concentration of 1μg/ml. Protease and leukotriene levels are assessed at 24 and 36 hourtime points.

The formulations of antibodies of the present invention thatimmunospecifically bind to an IL-9 polypeptide may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of an IL-9 polypeptideor may be specific for both an IL-9 polypeptide as well as for aheterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., International publications WO 93/17715, WO92/08802, WO 91/00360, and WO 92/05793; Tutt, et al., J. Immunol.147:60-69(1991); U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648,5,573,920, and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553(1992).

The present invention provides for antibodies that have a high bindingaffinity for an IL-9 polypeptide. In a specific embodiment, an antibodythat immunospecifically binds to an IL-9 polypeptide has an associationrate constant or k_(on) rate

$\left( {{{antibody}({Ab})} + {{{antigen}({Ag})}\overset{k_{on}}{\longrightarrow}{Ab}} - {Ag}} \right)$of at least 10⁵ M⁻¹s⁻¹, at least 1.5×10⁵ M⁻¹s⁻¹, at least 2×10⁵ M⁻¹s⁻¹,at least 2.5×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶M⁻¹s⁻¹, atleast 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or atleast 10⁸ M⁻¹s⁻¹, or 10⁵-10⁸M⁻¹s⁻, 1.5×10⁵ M⁻¹s⁻¹, 1×10⁷ M⁻¹s⁻¹,2×10⁵−1×10⁶M⁻¹s⁻¹, or 4.5×10⁵×10⁷ M⁻¹s⁻¹. In a preferred embodiment, anantibody that immunospecifically binds to an IL-9 polypeptide has ak_(on) of at least 2×10⁵ M⁻¹s⁻¹, at least 2.5×10⁵ M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least 10⁶M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, atleast 5×10⁷M⁻¹s⁻¹, or at least 10⁸ M⁻¹ s⁻¹ as determined by a BIAcoreassay and the antibody neutralizes human IL-9 in the microneutralizationassay as described herein. In a preferred embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide has a k_(on) of at most10⁸ M⁻¹s⁻¹, at most 10⁹ M⁻¹s⁻¹, at most 10¹⁰ M⁻¹s⁻¹, at most 10¹¹M⁻¹s⁻¹, or at most 10¹² M⁻¹s⁻¹ as determined by a BIAcore assay and theantibody neutralizes human IL-9 in the microneutralization assay asdescribed herein. In accordance with these embodiments, such antibodiesmay comprise a VH domain and/or a VL domain of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4 or a VH CDR and/or a VL CDR of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4.

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide has a k_(off) rate

$\left( {{{antibody}({Ab})} + {{{antigen}({Ag})}\overset{k_{off}}{\longleftarrow}{Ab}} - {Ag}} \right)$of less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁴ s⁻¹, lessthan 2×10⁴ s⁻¹, less than 5×10⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹,less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹, or10⁻³−10¹⁰ s⁻¹, 10⁻⁴-10⁻⁸ s⁻¹, or 10⁻⁵-10⁻⁸ s⁻¹. In a preferredembodiment, an antibody that immunospecifically binds to an IL-9polypeptide has a k_(off) of 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, lessthan 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹ as determined by a BIAcore assayand the antibody neutralizes human IL-9 in the microneutralization assaydescribed herein. In another preferred embodiment, an antibody thatimmunospecifically binds to an IL-9 polypeptide has a k_(off) of greaterthan 10⁻¹³ s⁻¹, greater than 10⁻¹² s⁻¹, greater than 10⁻¹¹ s⁻¹, greaterthan 10⁻¹⁰ s⁻¹, greater than 10⁻⁹ s⁻¹, or greater than 10⁻⁸ s-⁻¹. Inaccordance with these embodiments, such antibodies may comprise a VHdomain and/or a VL domain of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9,7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4, or a VH CDRand/or a VL CDR of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or 7F3com-3D4.

In another embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide has an affinity constant or K_(a) (k_(on)/k_(off)) ofat least 10² M⁻¹, at least 5×10² M⁻¹, at least 10³ M⁻¹, at least 5×10³M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least5×10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, atleast 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹,at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, at least10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹,at least 10¹³ M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹, or 10²−5×10⁵ M⁻¹,10⁴−1×10¹⁰ M⁻¹, or 10⁵−1×10⁸ M⁻¹. In another embodiment, an antibodythat immunospecificaly binds to an IL-9 polypeptide has a K_(a) of atmost 10¹¹ M⁻¹, at most 5×10¹¹M⁻¹, at most 10¹² M⁻¹, at most 5×10¹² M⁻¹,at most 10¹³ M⁻¹, at most 5×10¹³ M⁻¹, at most 10¹⁴ M⁻¹, or at most5×10¹⁴ M⁻¹. In another embodiment, an antibody that immunospecificallybinds to an IL-9 polypeptide has a dissociation constant or K_(d)(k_(off)/k_(on)) of less than 10⁻⁵ M, less than 5×10⁻⁵ M, less than 10⁻⁶M, less than 5×10⁻⁶ M, less than 10⁻⁷ M, less than 5×10⁻⁷ M, less than10⁻⁸ M, less than 5×10⁻⁸ M, less than 10⁻⁹ M, less than 5×10⁻⁹ M, lessthan 10⁻¹⁰ M, less than 5×10⁻¹⁰ M, less than 10¹¹ M, less than 5×10¹¹ M,less than 10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M, less than5×10⁻¹³ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁵ M, orless than 5 ×10⁻¹⁵ M or 10⁻² M−5×10⁻⁵ M, 10⁻⁶-10⁻¹⁵ M, or 10⁻⁸-10⁻¹⁴ M.In a preferred embodiment, an antibody that immunospecifically binds toan IL-9 polypeptide has a K_(d) of less than 10⁻⁹ M, less than 5×10⁻⁹ M,less than 10⁻¹⁰ M, less than 5×10⁻¹⁰ M, less than 1×10⁻¹¹ M, less than5×10⁻¹¹ M, less than 1×10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M,less than 5×10⁻¹³ M or less than 1×10⁻¹⁴ M, or 10⁻⁹ M-10⁻¹ ⁴ M asdetermined by a BIAcore assay and the antibody neutralizes human IL-9 inthe microneutralization assay described herein. In another preferredembodiment, an antibody that immunospecifically binds to an IL-9polypeptide has a K_(d) of greater than 10⁻⁹ M, greater than 5×10⁻⁹ M,greater than 10⁻¹⁰ M, greater than 5×10⁻¹⁰ M, greater than 10⁻¹ M,greater than 5×10⁻¹¹ M, greater than 10⁻¹² M, greater than 5×10⁻¹² M,greater than 6×10⁻¹² M, greater than 10⁻¹³ M, greater than 5×10⁻¹³ M,greater than 10⁻¹⁴ M, greater than 5×10⁻¹⁴ M or greater than 10⁻⁹M-10⁻¹⁴ M. In accordance with these embodiments, such antibodies maycomprise a VH domain and/or a VL domain of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4, or a VH CDR and/or a VL CDR of 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5 or7F3com-3D4.

In certain embodiments, formulations of the antibodies of the inventiondo not include antibodies known in the art that immunospecifically bindto an IL-9 polypeptide. Non-limiting examples of known antibodies thatimmunospecifically bind to an IL-9 polypeptide include 4D4, 4D4 H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5 or 7F3com-3D4.

In specific embodiments, formulations of antibodies of the inventionbind antigenic epitope-bearing peptides and polypeptides of IL-9, andsaid antigenic epitope-bearing peptides and polypeptides comprise orconsist of an amino acid sequence of at least 4, at least 5, at least 6,at least 7, more preferably at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least20, at least 25, at least 30, at least 40, at least 50 contiguous aminoacid residues, and, preferably, between about 15 to about 30 contiguousamino acids of IL-9 found in any species. Preferred polypeptidescomprising immunogenic or antigenic epitopes are at least 8, at least10, at least 15, at least 20, at least 25, at least at least 30, or atleast 35 amino acid residues in length.

IL-9 epitope-bearing peptides, polypeptides, and fragments thereof maybe produced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids,” Proc. Natl. Acad. Sci. USA 82:5 13 1-5 135;this “Simultaneous Multiple. Peptide Synthesis (SMPS)” process isfurther described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

The present invention provides formulations of peptides, polypeptidesand/or proteins comprising one or more variable or hypervariable regionsof the antibodies described herein. Preferably, peptides, polypeptidesor proteins comprising one or more variable or hypervariable regions ofantibodies of the invention further comprise a heterologous amino acidsequence. In certain embodiments, such a heterologous amino acidsequence comprises at least 5 contiguous amino acid residues, at least10 contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 30 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 75 contiguous amino acidresidues, at least 100 contiguous amino acid residues or more contiguousamino acid residues. Such peptides, polypeptides and/or proteins may bereferred to as fusion proteins.

In a specific embodiment, formulations of peptides, polypeptides orproteins comprising one or more variable or hypervariable regions of theantibodies of the invention are 10 amino acid residues, 15 amino acidresidues, 20 amino acid residues, 25 amino acid residues, 30 amino acidresidues, 35 amino acid residues, 40 amino acid residues, 45 amino acidresidues, 50 amino acid residues, 75 amino acid residues, 100 amino acidresidues, 125 amino acid residues, 150 amino acid residues or more aminoacid residues in length. In certain embodiments, peptides, polypeptides,or proteins comprising one or more variable or hypervariable regions ofan antibody of the invention immunospecifically bind to an IL-9polypeptide. In other embodiments, peptides, polypeptides, or proteinscomprising one or more variable or hypervariable regions of an antibodyof the invention do not immunospecifically bind to an IL-9 polypeptide.

In a specific embodiment, the present invention provides formulations ofpeptides, polypeptides and/or proteins comprising a VH domain and/or VLdomain of one of the antibodies described herein (see Table 1, supra).In a preferred embodiment, the present invention provides peptides,polypeptides and/or proteins comprising one or more CDRs having theamino acid sequence of any of the CDRs listed in Table 1, supra. Inaccordance with these embodiments, the peptides, polypeptides orproteins may further comprise a heterologous amino acid sequence.

Peptides, polypeptides or proteins comprising one or more variable orhypervariable regions have utility, e.g., in the production ofanti-idiotypic antibodies which in turn may be used to prevent, treat,and/or ameliorate one or more symptoms associated with a disease ordisorder (e.g., an autoimmune disorder, an inflammatory disorder, aproliferative disorder or an infection (preferably, a respiratoryinfection)). The anti-idiotypic antibodies produced can also be utilizedin immunoassays, such as, e.g., ELISAs, for the detection of antibodieswhich comprise a variable or hypervariable region contained in thepeptide, polypeptide or protein used in the production of theanti-idiotypic antibodies.

5.1.1.1. Antibodies Having Increased Half-Lives That ImmunospecificallyBind to an IL-9 Polypeptide

The present invention provides for formulations of antibodies andantibody fragments that immunospecifically bind to an IL-9 polypeptidewhich have a extended half-life in vivo. In particular, the presentinvention provides formulations of antibodies and antibody fragmentsthat immunospecifically bind to an IL-9 polypeptide which have ahalf-life in an animal, preferably a mammal and most preferably a human,of greater than 3 days, greater than 7 days, greater than 10 days,preferably greater than 15 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months.

To prolong the serum circulation of antibodies (e.g., monoclonalantibodies and single chain antibodies) or antibody fragments (e.g., Fabfragments) in vivo, for example, inert polymer molecules such as highmolecular weight polyethyleneglycol (PEG) can be attached to theantibodies (including antibody fragments thereof) with or without amultifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of the antibodies or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by size-exclusion or by ion-exchange chromatography.PEG-derivatized antibodies (including antibody fragments thereof) can betested for binding activity as well as for in vivo efficacy usingmethods known to those of skill in the art, for example, by immunoassaysdescribed herein.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge-Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375, each of whichis incorporated herein by reference in its entirety.

Further, antibodies (including antibody fragments thereof) can beconjugated to albumin in order to make the antibody (including antibodyfragment thereof) more stable in vivo or have a longer half life invivo. The techniques are well known in the art, see e.g., InternationalPublication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and EuropeanPatent No. EP 413, 622, all of which are incorporated herein byreference.

5.1.1.2. Antibody Conjugates

The present invention provides formulations of antibodies (includingantibody fragments thereof) that immunospecifically binds to an IL-9polypeptide recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous protein orpolypeptide (or fragment thereof, preferably to a polypeptide of atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90 or at least 100 amino acids)to generate fusion proteins. In particular, the invention providesformulations of fusion proteins comprising an antigen-binding fragmentof an antibody described herein (e.g., a Fab fragment, Fd fragment, Fvfragment, F(ab)₂ fragment, a VH domain, a VH CDR, a VL domain or a VLCDR) and a heterologous protein, polypeptide, or peptide. Preferably,the heterologous protein, polypeptide, or peptide that the antibody(including antibody fragments thereof) is fused to is useful fortargeting the antibody to respiratory epithelial cells, mast cells,neutrophils, eosinophils, B cells, macrophages, or activated T cells.For example, an antibody that immunospecifically binds to a cell surfacereceptor expressed by a particular cell type (e.g., a respiratoryepithelial cell, a mast cell, a neutrophil, an eosinophil, a B cell, amacrophage, or an activated T cell) may be fused or conjugated to anantibody (including antibody fragment thereof) of the invention. In aspecific embodiment, an antibody that immunospecifically binds to anIL-9 polypeptide is fused or conjugated to an anti-stem cell factor oran anti-kit ligand. Methods for fusing or conjugating proteins,polypeptides, or peptides to an antibody (including antibody fragmentthereof) are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EuropeanPatent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; andVil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (saidreferences are incorporated herein by reference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al, 1999, J. Mol. Biol. 287:265-76;and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of thesepatents and publications are hereby incorporated by reference in itsentirety). Antibodies (including antibody fragments thereof), or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody (including antibody fragment thereof) thereof thatimmunospecifically binds to an IL-9 polypeptide may be recombined withone or more components, motifs, sections, parts, domains, fragments,etc. of one or more heterologous molecules.

Moreover, the antibodies (including antibody fragments thereof) can befused to marker sequences, such as a peptide to facilitate purification.In preferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin (“HA”) tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,1984, Cell 37:767), and the “flag” tag.

In other embodiments, antibodies of the present invention or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder (e.g., anautoimmune disorder, an inflammatory disorder, a proliferative disorder,or an infection (preferably, a respiratory infection)) as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I,), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In,),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶R, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵ Zn, ⁸⁵ Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using variouspositron emission tomographies, and noradioactive paramagnetic metalions.

The present invention further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Therapeuticmoieties include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine); alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP), and cisplatin);anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithranycin, and anthramycin (AMC)); Auristatin molecules(e.g., auristatin PHE, bryostatin 1, and solastatin 10; see Woyke etal., Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al.,Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al.,Anticancer Drugs 12:735-40 (2001), Wall et al., Biochem. Biophys. Res.Commun. 266:76-80 (1999), Mohammad et al., Int. J. Oncol. 15:367-72(1999), all of which are incorporated herein by reference); hormones(e.g., glucocorticoids, progestins, androgens, and estrogens),DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinaseinhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al.,Clin Cancer Res. 8(7):2167-76 (2002)); cytotoxic agents (e.g.,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof and those compounds disclosed in U.S. Pat. Nos.6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459); farnesyl transferase inhibitors (e.g.,R115777, BMS-214662, and those disclosed by, for example, U.S. Pat. Nos.6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305);topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38;topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-895 If; IST-622;rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022;TAN-1518A; TAN-1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506;and rebeccamycin); bulgarein; DNA minor groove binders such as Hoeschtdye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; bisphosphonates (e.g., alendronate,cimadronte, clodronate, tiludronate, etidronate, ibandronate,neridronate, olpandronate, risedronate, piridronate, pamidronate,zolendronate) HMG-CoA reductase inhibitors, (e.g., lovastatin,simvastatin, atorvastatin, pravastatin, fluvastatin, statin,cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin); antisenseoligonucleotides (e.g., those disclosed in the U.S. Pat. Nos. 6,277,832,5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminaseinhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine);ibritumomab tiuxetan (ZEVALIN®); tositumomab (BEXXAR®)) andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, a-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, TNF-β,AIM I (see, International Publication No. WO 97/33899), AIM II (see,International Publication No. WO 97/34911), Fas Ligand (Takahashi etal., 1994, J. Immunol., 6:1567-1574), and VEGF (see, InternationalPublication No. WO 99/23105), an anti-angiogenic agent, e.g.,angiostatin, endostatin or a component of the coagulation pathway (e.g.,tissue factor); or, a biological response modifier such as, for example,a lymphokine (e.g., interferon gamma (“IFN-γ”), interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”),interleuking-7 (“IL-7”), interleukin-10 (“IL-10”), interleukin-12(“IL-12”), interleukin-15 (“IL-15”), interleukin-23 (“IL-23”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)), or a coagulation agent (e.g., calcium,vitamin K, tissue factors, such as but not limited to, Hageman factor(factor XII), high-molecular-weight kininogen (HMWK), prekallikrein(PK), coagulation proteins-factors II (prothrombin), factor V, XIIa,VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid fibrinopeptides A and Bfrom the α and β chains of fibrinogen, fibrin monomer). In a specificembodiment, an antibody that immunospecifically binds to an IL-9polypeptide is conjugated with a leukotriene antagonist (e.g.,montelukast, zafirlukast, pranlukast, and zyleuton).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alph-emiters such as ²¹³Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹³¹In, ¹³¹L, ¹³¹y, ¹³¹Ho, ¹³¹Sm, to polypeptides orany of those listed supra. In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999,Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med.Biol. 26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorp, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated to an antibody thatimmunospecifically binds to an IL-9 polypeptide or fragment thereofshould be chosen to achieve the desired prophylactic or therapeuticeffect(s) for a particular disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof, in a subject. A clinician or other medicalpersonnel should consider the following when deciding on whichtherapeutic moiety or drug to conjugate to an antibody thatimmunospecifically binds to an IL-9 polypeptide or fragment thereof: thenature of the disease, the severity of the disease, and the condition ofthe subject.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

The therapeutic moiety or drug conjugated to an antibody (includingantibody fragment thereof) that immunospecifically binds to an IL-9polypeptide should be chosen to achieve the desired prophylactic ortherapeutic effect(s) for a particular disorder in a subject. Aclinician or other medical personnel should consider the following whendeciding on which therapeutic moiety or drug to conjugate to an antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide: the nature of the disease, the severity of thedisease, and the condition of the subject.

5.2. Method of Preparing the Antibody Formulations

The present invention provides methods for preparing liquid formulationsof antibodies or derivatives, analogues, or fragments thereof thatimmunospecifically bind to an IL-9 polypeptide. FIG. 16 is a schematicdiagram showing the outline for preparing purified anti-IL-9 antibodies.The methods for preparing liquid formulations of the present inventioncomprise: purifying the antibody (including antibody fragment thereof)from conditioned medium (either single lots or pooled lots of medium)and concentrating a fraction of the purified antibody (includingantibody fragment thereof) to a final concentration of from about 15mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml,about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about100 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 250mg/ml, or about 300 mg/ml. Conditioned medium containing the antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide is subjected to CUNO filtration and the filteredantibody is subjected to HS50 cation exchange chromatography. Thefraction from the HS50 cation exchange chromatography is then subjectedto rProtein A affinity chromatography followed by low pH treatment.Following low pH treatment, the antibody (including antibody fragmentthereof) fraction is subject to super Q 650 anion exchangechromatography and then nanofiltration. The fraction of the antibody(including antibody fragment thereof) obtained after nanofiltration isthen subjected to diafiltration to concentrate the antibody (includingantibody fragment thereof) fraction into the formulation buffer usingthe same membrane. For a detailed description for preparation of theantibody formulations, see Section 6, infra.

The formulation buffer of the present invention comprises histidine at aconcentration ranging from about 1 mM to about 100 mM, about 5 mM toabout 50 mM, about 10 mM to about 30 mM, or about 10 mM to about 25 mM.In a specific embodiment, the formulation buffer of the presentinvention comprises histidine at a concentration of about 10 mM, about12 mM, about 15 mM, about 20 mM or about 25 mM. The formulations mayfurther comprise glycine at a concentration of less than 150 mM, lessthan 100 mM, less than 75 mM, less than 50 mM, less than 10 mM, lessthan 3.0 mM, or less than 2.0 mM. The amount of glycine in theformulation should not cause a significant buffering in order to avoidantibody precipitation at its isoelectric point. The pH of theformulation may range from about 5.0 to about 7.0, preferably about 5.5to about 6.5, more preferably about 5.8 to about 6.2, and mostpreferably about 6.0. To obtain an appropriate pH for a particularantibody, it is preferable that histidine (and glycine, if added) isfirst dissolved in water to obtain a buffer solution with higher pH thanthe desired pH and then the pH is brought down to the desired level byadding HCl. This way, the formation of additional inorganic salts (e.g.,formation of NaCl when, for example, histidine hydrochloride is used ashistidine and pH is raised to a desired level by adding NaOH) can beavoided.

The liquid formulations of the present invention can be prepared as unitdosage forms by preparing a vial containing an aliquot of the liquidformulation for a one-time use. For example, a unit dosage per vial maycontain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15ml, or 20 ml of different concentrations of an antibody (includingantibody fragment thereof) that immunospecifically binds to an IL-9polypeptide ranging from about 15 mg/ml to about 300 mg/ml. Ifnecessary, these preparations can be adjusted to a desired concentrationby adding a sterile diluent to each vial. In a preferred embodiment, theliquid formulations of the present invention are formulated into singledose vials as a sterile liquid that contains 10 mM histidine buffer atpH 6.0 and 150 mM sodium chloride. Each 1.0 mL of solution contains 100mg of the antibody (including antibody fragment thereof), 1.6 mg ofhistidine and 8.9 mg of sodium chloride in water. During themanufacturing process, the pH of the formulation buffer is adjusted to6.0 using hydrochloric acid. In a preferred embodiment, the antibody(including antibody fragment thereof) of the invention is supplied at100 mg/ml in 3 cc USP Type I borosilicate amber vials (WestPharmaceutical Services—Part No. 6800-0675). The target fill volume is1.2 mL.

The liquid formulations of the present invention may be sterilized byvarious sterilization methods, including sterile filtration, radiation,etc. In a most preferred embodiment, the difiltrated antibodyformulation is filter-sterilized with a presterilized 0.2 micron filter.Sterilized liquid formulations of the present invention may beadministered to a subject to prevent, treat or ameliorate a disease ordisorder (e.g., a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection)) or one or moresymptoms thereof.

Although the invention is directed to liquid non-lyophilizedformulations, it should be noted for the purpose of equivalents that theformulations of the invention may be lyophilized if desired. Thus, theinvention encompasses lyophilized forms of the formulations of theinvention although such lyophilized formulations are not preferred.

5.3. Methods of Preparing Antibodies

The antibodies (including antibody fragments thereof) thatimmunospecifically bind to an antigen can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

Polyclonal antibodies immunospecific for an antigen can be produced byvarious procedures well-known in the art. For example, a human antigencan be administered to various host animals including, but not limitedto, rabbits, mice, rats, etc. to induce the production of seracontaining polyclonal antibodies specific for the human antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981), and Harlow et al., UsingAntibodies: A laboratory Manual, Cold Spring Harbor Laboratory Press(1999) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with a non-murine antigen and once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. Additionally, a RIMMS (repetitive immunizationmultiple sites) technique can be used to immunize an animal (Kilpatracket al., 1997, Hybridoma 16:381-9, incorporated herein by reference inits entirety). The hybridoma clones are then assayed by methods known inthe art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

The present invention provides methods of generating monoclonalantibodies as well as antibodies produced by the method comprisingculturing a hybridoma cell secreting an antibody of the inventionwherein, preferably, the hybridoma is generated by fusing splenocytesisolated from a mouse immunized with a non-murine antigen with myelomacells and then screening the hybridomas resulting from the fusion forhybridoma clones that secrete an antibody able to bind to the antigen.

Antibody fragments which recognize specific particular epitopes may begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments of the invention may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the CH1 domain of the heavy chain. Further,the antibodies of the present invention can also be generated usingvarious phage display methods known in the art.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of affected tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector. The vector is electroporated in E. coli and the E. coliis infected with helper phage. Phage used in these methods are typicallyfilamentous phage including fd and M13 and the VH and VL domains areusually recombinantly fused to either the phage gene III or gene VIII.Phage expressing an antigen binding domain that binds to a particularantigen can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J.Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J.Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,1994, Advances in Immunology 57:191-280; International application No.PCT/GB91/O1134; International Publication Nos. WO 90/02809, WO 91/10737,WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, andWO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,5,516,637, 5,780,225, 5,658,727, 5,733,743, 5,969,108, 6,33,187,5,824,520, and 5,702,892; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)2 fragments can also be employed using methods knownin the art such as those disclosed in PCT publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995,AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (saidreferences incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing a VHconstant region, e.g., the human gamma 4 constant region, and the PCRamplified VL domains can be cloned into vectors expressing a VL constantregion, e.g., human kappa or lamba constant regions. Preferably, thevectors for expressing the VH or VL domains comprise an EF-1α promoter,a secretion signal, a cloning site for the variable domain, constantdomains, and a selection marker such as neomycin. The VH and VL domainsmay also cloned into one vector expressing the necessary constantregions. The heavy chain conversion vectors and light chain conversionvectors are then co-transfected into cell lines to generate stable ortransient cell lines that express full-length antibodies, e.g., IgG,using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use humanized antibodiesor chimeric antibodies. Completely human antibodies and humanizedantibodies are particularly desirable for therapeutic treatment of humansubjects. Human antibodies can be made by a variety of methods known inthe art including phage display methods described above using antibodylibraries derived from human immunoglobulin sequences. See also U.S.Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then be bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Methodsfor producing chimeric antibodies are known in the art. See e.g.,Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214;Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat.Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, which areincorporated herein by reference in their entirety.

A humanized antibody is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immuoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)₂, Fabc, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both the lightchain as well as at least the variable domain of a heavy chain. Theantibody also may include the CH1, hinge, CH2, CH3, and CH4 regions ofthe heavy chain. The humanized antibody can be selected from any classof immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and anyisotype, including IgG₁, IgG₂, IgG₃ and IgG₄. Usually the constantdomain is a complement fixing constant domain where it is desired thatthe humanized antibody exhibit cytotoxic activity, and the class istypically IgG₁. Where such cytotoxic activity is not desirable, theconstant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art. The framework and CDR regions of ahumanized antibody need not correspond precisely to the parentalsequences, e.g., the donor CDR or the consensus framework may bemutagenized by substitution, insertion or deletion of at least oneresidue so that the CDR or framework residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parentalframework and CDR sequences, more often 90%, and most preferably greaterthan 95%. Humanized antibody can be produced using variety of techniquesknown in the art, including but not limited to, CDR-grafting (EuropeanPatent No. EP 239,400; International publication No. WO 91/09967; andU.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering orresurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan,1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994,Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO9317105, Tan et al., J. Immunol. 169:1119-25 (2002), Caldas et al.,Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267-79(2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska etal., Protein Eng. 9(10):895-904 (1996), Couto et al., Cancer Res. 55 (23Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22(1995), Sandhu J S, Gene 150(2):409-10 (1994), and Pedersen et al., J.Mol. Biol. 235(3):959-73 (1994). Often, framework residues in theframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions (see, e.g., Queen et al., U.S. Pat. No.5,585,089; and Riechmann et al., 1988, Nature 332:323, which areincorporated herein by reference in their entireties).

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well-known in the art. See Riechmannet al., 1999, J. Immuno. 231:25-38; Nuttall et al., 2000, Curr. Pharm.Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302;U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94/04678,WO 94/25591, and WO 01/44301, each of which is incorporated herein byreference in its entirety.

Further, the antibodies that immunospecifically bind to an antigen(e.g., an IL-9 polypeptide) can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” an antigen using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1989,FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438).

5.3.1. Recombinant Expression of an Antibody

Recombinant expression of an antibody contained in a formulation of theinvention (e.g., a heavy or light chain of an antibody of the inventionor a fragment thereof or a single chain antibody of the invention) thatimmunospecifically binds to an IL-9 polypeptide requires construction ofan expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule, heavy orlight chain of an antibody, or fragment thereof (preferably, but notnecessarily, containing the heavy or light chain variable domain) of theinvention has been obtained, the vector for the production of theantibody molecule may be produced by recombinant DNA technology usingtechniques well-known in the art. Thus, methods for preparing a proteinby expressing a polynucleotide containing an antibody encodingnucleotide sequence are described herein. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, a heavy or light chain of an antibody, a heavy or light chainvariable domain of an antibody (including antibody fragment thereof), ora heavy or light chain CDR, operably linked to a promoter. Such vectorsmay include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication No. WO 86/05807;International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464)and the variable domain of the antibody may be cloned into such a vectorfor expression of the entire heavy, the entire light chain, or both theentire heavy and light chains.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention or fragments thereof, or a heavy or light chain thereof,or fragment thereof, or a single chain antibody of the invention,operably linked to a heterologous promoter. In preferred embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains may be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule of the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, NS0, and 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).Preferably, bacterial cells such as Escherichia coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,Bio/Technology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding antibodies of the invention, derivative,analog, or fragment thereof which immunospecifically bind to an IL-9polypeptide or fragments thereof is regulated by a constitutivepromoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO12:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan andAnderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH11(5):155-2 15); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley& Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1,which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler,1980, Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for theheavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

5.4. Methods of Monitoring the Stability and Aggregation of AntibodyFormulations

There are various methods available for assessing the stability ofprotein formulations, including antibody formulations, based on thephysical and chemical structures of the proteins as well as on theirbiological activities. For example, to study denaturation of proteins,methods such as charge-transfer absorption, thermal analysis,fluorescence spectroscopy, circular dichroism, NMR, and HPSEC, areavailable. See, for example, Wang et al., 1988, J. of Parenteral Science& Technology 42(Suppl):S4-S26.

The rCGE and HPSEC are the most common and simplest methods to assessthe formation of protein aggregates, protein degradation, and proteinfragmentation. Accordingly, the stability of the liquid formulations ofthe present invention may be assessed by these methods.

For example, the stability of the liquid formulations of the presentinvention may be evaluated by HPSEC or rCGE, wherein the percent area ofthe peaks represents the non-degraded antibody or non-degraded antibodyfragments. In particular, approximately 250 μg of the antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide (approximately 25 μl of a liquid formulationcomprising 10 mg/ml said antibody or antibody fragment) is injected ontoa TosoH Biosep TSK G3000SW_(XL) column (7.8 mm×30 cm) fitted with a TSKSW×1 guard column (6.0 mm CX 4.0 cm). The antibody (including antibodyfragment thereof) is eluted isocratically with 0.1 M disodium phosphatecontaining 0.1 M sodium sulfate and 0.05% sodium azide, at a flow rateof 0.8 to 1.0 ml/min. Eluted protein is detected using UV absorbance at280 nm. 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F322D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 (or any other antibody thatis in a formulation of the invention) reference standards are run in theassay as controls, and the results are reported as the area percent ofthe product monomer peak compared to all other peaks excluding theincluded volume peak observed at approximately 12 to 14 minutes. Peakseluting earlier than the monomer peak are recorded as percent aggregate.

The liquid formulations of the present invention exhibit low toundetectable levels of aggregation as measured by HPSEC or rCGE, thatis, no more than 5%, no more than 4%, no more than 3%, no more than 2%,no more than 1%, and most preferably no more than 0.5% aggregate byweight protein, and low to undetectable levels of fragmentation, thatis, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% orhigher, or 99% or higher, or 99.5% or higher of the total peak area inthe peak(s) representing intact antibodies (including antibody fragmentsthereof). In the case of SDS-PAGE, the density or the radioactivity ofeach band stained or labeled with radioisotope can be measured and the %density or % radioactivity of the band representing non-degradedantibodies (including antibody fragments thereof) can be obtained.

The stability of the liquid formulations of the present invention can bealso assessed by any assays which measure the biological activity of theantibody in the formulation. The biological activities of antibodiesinclude, but are not limited to, antigen-binding activity,complement-activation activity, Fc-receptor binding activity, and soforth. Antigen-binding activity of the antibodies (including antibodyfragments thereof) can be measured by any method known to those skilledin the art, including but not limited to ELISA, radioimmunoassay,Western blot, and the like. Complement-activation activity can bemeasured by a C3a/C4a assay in the system where the antibody whichimmunospecifically binds to an IL-9 polypeptide is reacted in thepresence of the complement components with the cells expressing the anIL-9 polypeptide. Also see Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)(incorporated by reference herein in its entirety). An ELISA basedassay, e.g., may be used to compare the ability of an antibody(including antibody fragments thereof) to immunospecifically bind to anIL-9 polypeptide to 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 (or any otherantibody that is in a formulation of the invention) reference standards.In this assay, referred to as the VnR Binding ELISA, plates are coatedwith an isolated IL-9 polypeptide and the binding signal of a setconcentration of 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10,7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 reference standards iscompared to the binding signal of the same concentration of a testantibody (including antibody fragment thereof).

The purity of the liquid antibody formulations of the invention may bemeasured by any method well-known to one of skill in the art such as,e.g., HPSEC. The sterility of the liquid antibody formulations may beassessed as follows: sterile soybean-casein digest medium and fluidthioglycollate medium are inoculated with a test liquid antibodyformulation by filtering the liquid antibody formulation through asterile filter having a nominal porosity of 0.45 μm. When using theSterisure™ or Steritest™ method, each filter device is asepticallyfilled with approximately 100 ml of sterile soybean-casein digest mediumor fluid thioglycollate medium. When using the conventional method, thechallenged filter is aseptically transferred to 100 ml of sterilesoybean-casein digest medium or fluid thioglycollate medium. The mediaare incubated at appropriate temperatures and observed three times overa 14 day period for evidence of bacterial or fungal growth.

5.5. Prophylactic and Therapeutic Utility of the Antibody Formulations

The present invention is also directed to antibody-based therapies whichinvolve administering to a subject, preferably a human, the liquidantibody formulations (or “antibody formulations” or “liquidformulations”) of the present invention for preventing, treating,managing or ameliorating a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof (see U.S. Provisional Appn. No. 60/477,801,filed Jun. 10, 2003, entitled “Methods of Preventing or TreatingRespiratory Conditions,” U.S. Provisional Appn. No. 60/462,307, filedApr. 11, 2003, entitled “Methods of Preventing or Treating RespiratoryConditions” and a U.S. patent application Ser. No. 10/823,810 filedconcurrently herewith on Apr. 12, 2004, entitled “Methods of Preventingor Treating Respiratory Conditions,” which are all incorporated byreference herein in their entireties). The liquid formulations of theinvention comprise an antibody (including antibody fragment thereof) atconcentrations of from about 15 mg/ml to about 300 mg/ml in a solutioncontaining histidine, which antibody (including antibody fragmentthereof) immunospecifically binds to an IL-9 polypeptide. The liquidformulations of the invention may comprise a single antibody (includingantibody fragment thereof) that immunospecifically binds to an IL-9polypeptide (e.g., 4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3,71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4). The liquidformulations of the invention may also comprise two or more antibodies(including antibody fragments thereof) that immunospecifically bind toan IL-9 polypeptide. In a specific embodiment, antibodies (includingantibody fragments thereof) included in such liquid formulations are4D4, 4D4 H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3,7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or fragments thereof. In analternative embodiment, antibodies (including antibody fragmentsthereof) included in such liquid formulations are not 4D4, 4D4 H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or fragments thereof. In yet another embodiment, theliquid formulations of the invention comprise an antibody (includingantibody fragment thereof) that immunospecifically binds to an IL-9polypeptide, and the antibody (including antibody fragment thereof) isalso conjugated to another moiety, including but not limited to, aheterologous protein, peptide or polypeptide, another antibody(including antibody fragment thereof), a marker sequence, a diagnosticagent, a therapeutic agent, a radioactive metal ion, and a solidsupport.

The liquid formulations of the present invention may be used locally orsystemically in the body as a therapeutic. Particularly, the liquidformulations of the invention may be used in the prevention, treatment,management and amelioration of a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof. The formulations of the invention can be usedto regulate the activity of cells expressing an IL-9R. In a specificembodiment, the formulations of the invention are used to regulatevarious activities of a body, including but not limited to, immunefunctions. The formulations of the present invention may also beadvantageously utilized in combination with one or more other therapies(e.g., one or more other prophylactic or therapeutic agents), preferablytherapies useful in the treatment, prevention, management oramelioration of a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof. When one or more other therapies (e.g., prophylacticor therapeutic agents) are used, they can be administered separately, inany appropriate form and by any suitable route. Therapeutic orprophylactic agents include, but are not limited to, small molecules,synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g.,DNA and RNA nucleotides including, but not limited to, antisensenucleotide sequences, triple helices, RNAi, and nucleotide sequencesencoding biologically active proteins, polypeptides or peptides)antibodies, synthetic or natural inorganic molecules, mimetic agents,and synthetic or natural organic molecules.

Any therapy (e.g., prophylactic or therapeutic agents) which is known tobe useful, or which has been used or is currently being used for theprevention, management, treatment, or amelioration of one or moresymptoms associated with a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), can beused in combination with the liquid antibody formulations of the presentinvention in accordance with the invention described herein. See, e.g.,Gilman et al., Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, Tenth Ed., McGraw-Hill, New York, 2001; The Merck Manualof Diagnosis and Therapy, Berkow, M. D. et al. (eds.), 17th Ed., MerckSharp & Dohme Research Laboratories, Rahway, N.J., 1999; and CecilTextbook of Medicine, 20th Ed., Bennett and Plum (eds.), W.B. Saunders,Philadelphia, 1996 for information regarding therapies, in particularprophylactic or therapeutic agents, which have been or are currentlybeing used for preventing, treating, managing, and/or amelioratingdiseases or disorders associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, diseases or disordersassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, autoimmune diseases,inflammatory diseases, proliferative diseases, or infections(preferably, respiratory infections), or one or more symptoms thereof.Examples of prophylactic and therapeutic agents include, but are notlimited to, immunomodulatory agents, anti-inflammatory agents (e.g.,adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide,flunisolide, fluticasone, triamcinolone, methlyprednisolone,prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids,non-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen,diclofenac, and COX-2 inhibitors), and leukotreine antagonists (e.g.,montelukast, methyl xanthines, zafirlukast, and zileuton),beta2-agonists (e.g., albuterol, biterol, fenoterol, isoetharie,metaproterenol, pirbuterol, salbutamol, terbutalin formoterol,salmeterol, and salbutamol terbutaline), anticholinergic agents (e.g.,ipratropium bromide and oxitropium bromide), sulphasalazine,penicillamine, dapsone, antihistamines, anti-malarial agents (e.g.,hydroxychloroquine), anti-viral agents, and antibiotics (e.g.,dactinomycin (formerly actinomycin), bleomycin, erythomycin, penicillin,mithramycin, and anthramycin (AMC)).

A liquid formulation of the invention may be administered to a mammal,preferably a human, concurrently with one or more other therapies (e.g.,one or more other prophylactic or therapeutic agents), preferablytherapies useful for the prevention, treatment, management oramelioration of a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof. The term “concurrently” is not limited to theadministration of prophylactic or therapeutic agents/therapies atexactly the same time, but rather it is meant that a liquid formulationof the invention and the other agent/therapy are administered to amammal in a sequence and within a time interval such that the antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide contained in the liquid formulation can act togetherwith the other agent/therapy to provide an increased benefit than ifthey were administered otherwise. For example, a liquid formulation ofthe invention and one or more other prophylactic or therapeutic agentsuseful for prevention, treatment, management or amelioration of adisease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof,may be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic or prophylactic effect.

In various embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents), preferably therapies useful for prevention,treatment, management or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, a liquid formulation of the invention and oneor more other therapies (e.g., one or more other prophylactic ortherapeutic agents), preferably therapies useful for prevention,treatment, management or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, areadministered within the same patient visit. In other embodiments, aliquid formulation of the invention and one or more other therapies(e.g., one or more other prophylactic or therapeutic agents), preferablytherapies useful for prevention, treatment, management or ameliorationof a disease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof,are administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart. In preferred embodiments, a liquid formulation of the inventionand one or more other therapies (e.g., prophylactic or therapeuticagents), preferably therapies useful for prevention, treatment,management or amelioration of a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof, are administered in a time frame where bothagents are still active. One skilled in the art would be able todetermine such a time frame by determining the half-life of theadministered agents.

In certain embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents), preferably therapies useful for prevention,treatment, management or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, are cyclicallyadministered to a subject. Cycling therapy involves the administrationof a first agent for a period of time, followed by the administration ofa second agent and/or third agent for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improves theefficacy of the treatment.

In certain embodiments, a liquid formulation of the invention and one ormore other therapies (e.g., one or more other prophylactic ortherapeutic agents), preferably therapies useful for prevention,treatment, management or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, areadministered in a cycle of less than about 3 weeks, about once every twoweeks, about once every 10 days or about once every week. One cycle cancomprise the administration of a therapeutic or prophylactic agent byinfusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

In other embodiments, liquid formulation of the invention and one ormore other therapies (e.g., prophylactic or therapeutic agents),preferably therapies useful for prevention, treatment, management oramelioration of a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof, are administered in metronomic dosing regimens, eitherby continuous infusion or frequent administration without extended restperiods. Such metronomic administration can involve dosing at constantintervals without rest periods. Typically the prophylactic ortherapeutic agents, in particular cytotoxic agents, are used at lowerdoses. Such dosing regimens encompass the chronic daily administrationof relatively low doses for extended periods of time. In preferredembodiments, the use of lower doses can minimize toxic side effects andeliminate rest periods. In certain embodiments, the prophylactic andtherapeutic agents are delivered by chronic low-dose or continuousinfusion ranging from about 24 hours to about 2 days, to about 1 week,to about 2 weeks, to about 3 weeks to about 1 month to about 2 months,to about 3 months, to about 4 months, to about 5 months, to about 6months.

In one embodiment, a liquid formulation of the invention is administeredin a dosing regimen that maintains the plasma concentration of theantibody (including antibody fragment thereof) immunospecific for anIL-9 polypeptide at a desirable level (e.g., about 0.1 to about 100μg/ml), which continuously blocks the an IL-9R activity. In a specificembodiment, the plasma concentration of the antibody (including antibodyfragment thereof) is maintained at 0.2 μg/ml, 0.5 μg/ml, 1 μg/ml, 2μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45μg/ml or 50 μg/ml. The plasma concentration that is desirable in asubject will vary depending on several factors, including but notlimited to, the nature of the disease or disorder, the severity of thedisease or disorder and the condition of the subject. Such dosingregimens are especially beneficial in prevention, treatment, managementand amelioration of a chronic disease or disorder.

In one embodiment, a liquid formulation of the invention is administeredto a subject with a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof using a dosing regimen that maintains the plasmaconcentration of the an antibody (including antibody fragment thereof)that immunospecifically binds to an IL-9 polypeptide at a level thatblocks at least 40%, preferably at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90% or at least 95% of IL-9R binding to an IL-9polypeptide. In a specific embodiment, the plasma concentration of thean antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide is maintained at about0.1 μg/ml to about 100 μg/ml in a subject with a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof.

In some embodiments, a liquid formulation of the invention isadministered intermittently to a subject, wherein the liquid formulationcomprises an antibody (including antibody fragment thereof) conjugatedto a moiety (e.g., a therapeutic agent or a toxin).

When used in combination with other therapies (e.g., prophylactic and/ortherapeutic agents) useful for prevention, treatment, management oramelioration of a disease or disorder associated with or characterizedby aberrant expression and/or activity of an IL-9 polypeptide, a diseaseor disorder associated with or characterized by aberrant expressionand/or activity of the IL-9R or one or more subunits thereof, anautoimmune disease, an inflammatory disease, a proliferative disease, oran infection (preferably, a respiratory infection), or one or moresymptoms thereof, the liquid formulations of the invention and the othertherapy can act additively or, more preferably, synergistically. Theinvention contemplates administration of a liquid formulation of theinvention in combination with other therapies (e.g., prophylactic ortherapeutic agents) preferably therapies useful for prevention,treatment, management or amelioration of a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof by the same ordifferent routes of administration, e.g., oral and parenteral. Incertain embodiments, when a liquid formulation of the invention isadministered concurrently with one or more therapies (e.g., prophylacticor therapeutic agents) that potentially produce adverse side effects(including, but not limited to, toxicity), the therapies (e.g.,prophylactic or therapeutic agents) can advantageously be administeredat a dose that falls below the threshold that the adverse side effect iselicited.

5.5.1. Cancer Treatment

The liquid formulations of the invention may be administered to asubject in need thereof to prevent, treat, manage or ameliorate a canceror one or more symptoms thereof. The liquid formulations of theinvention may also be administered in combination with one or more othertherapies, preferably therapies useful for the prevention, management ortreatment of cancer (including, but not limited to the prophylactic ortherapeutic agents listed in Section 5.5.1.1 hereinbelow) to a subjectin need thereof to prevent, treat, manage or ameliorate a cancer or oneor more symptoms thereof. In a specific embodiment, the inventionprovides a method of preventing, treating, managing or amelioratingcancer or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof a dose of a prophylacticallyor therapeutically effective amount of a liquid formulation of theinvention. In another embodiment, the invention provides a method ofpreventing, treating or ameliorating cancer or one or more symptomsthereof, said method comprising administering to a subject in needthereof a dose of a prophylactically or therapeutically effective amountof a liquid formulation of the invention and a dose of aprophylactically or therapeutically effective amount of one or moretherapies (e.g., prophylactic or therapeutic agents other thanantibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide).

The liquid formulations of the invention may be used as a first, second,third or fourth line cancer treatment. The invention provides methodsfor treating or ameliorating one or more symptoms of a cancer in asubject refractory to conventional therapies for such a cancer, saidmethods comprising administering to said subject a dose of aprophylactically or therapeutically effective amount of a liquidformulation of the invention. A cancer may be determined to berefractory to a therapy means when at least some significant portion ofthe cancer cells are not killed or their cell division arrested inresponse to the therapy. Such a determination can be made either in vivoor in vitro by any method known in the art for assaying theeffectiveness of treatment on cancer cells, using the art-acceptedmeanings of “refractory” in such a context. In a specific embodiment, acancer is refractory where the number of cancer cells has not beensignificantly reduced, or has increased.

The invention provides methods for managing, treating or amelioratingcancer or one or more symptoms thereof in a subject refractory toexisting single agent therapies for such a cancer, said methodscomprising administering to said subject a dose of a prophylactically ortherapeutically effective amount of a liquid formulation of theinvention and a dose of a prophylactically or therapeutically effectiveamount of one or more therapies (e.g., prophylactic or therapeuticagents) other than antibodies (including antibody fragments thereof)that immunospecifically bind to an IL-9 polypeptide. The invention alsoprovides methods for managing, treating or ameliorating cancer byadministering a liquid formulation of the invention in combination withany other treatment (e.g., radiation therapy, chemotherapy or surgery)to patients who have proven refractory to other treatments but are nolonger on these treatments. The invention also provides methods for themanagement or treatment of a patient having cancer and immunosuppressedby reason of having previously undergone other cancer therapies. Theinvention also provides alternative methods for the management,treatment or amelioration of cancer or one or more symptoms thereof,where chemotherapy, radiation therapy, hormonal therapy, and/orbiological therapy/immunotherapy has proven or may prove too toxic,i.e., results in unacceptable or unbearable side effects, for thesubject being treated. Further, the invention provides methods forpreventing the recurrence of cancer in patients that have been treatedand have no disease activity by administering a liquid formulation ofthe invention.

Cancers that can be treated by the methods encompassed by the inventioninclude, but are not limited to, neoplasms, tumors, metastases, or anydisease or disorder characterized by uncontrolled cell growth. Thecancer may be a primary or metastatic cancer. The cancer may or may notexpress an IL-9R. Specific examples of cancers that can be treated bythe methods encompassed by the invention include, but are not limitedto, cancer of the head, neck, eye, mouth, throat, esophagus, chest,bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney,liver, pancreas, and brain. Additional cancers include, but are notlimited to, the following: leukemias such as but not limited to, acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemias such asmyeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemialeukemias and myelodysplastic syndrome, chronic leukemias such as butnot limited to, chronic myelocytic (granulocytic) leukemia, chroniclymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomassuch as but not limited to Hodgkin's disease, non-Hodgkin's disease;multiple myelomas such as but not limited to smoldering multiplemyeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cellleukemia, solitary plasmacytoma and extramedullary plasmacytoma;Waldenström's macroglobulinemia; monoclonal gammopathy of undeterminedsignificance; benign monoclonal gammopathy; heavy chain disease; bonecancer and connective tissue sarcomas such as but not limited to bonesarcoma, myeloma bone disease, osteosarcoma, chondrosarcoma, Ewing'ssarcoma, Paget's disease of bone, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease (includingjuvenile Paget's disease), and inflammatory breast cancer; adrenalcancer such as but not limited to pheochromocytom and adrenocorticalcarcinoma; thyroid cancer such as but not limited to papillary orfollicular thyroid cancer, medullary thyroid cancer and anaplasticthyroid cancer; pancreatic cancer such as but not limited to,insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secretingtumor, and carcinoid or islet cell tumor; pituitary cancers such as butlimited to Cushing's disease, prolactin-secreting tumor, acromegaly, anddiabetes insipius; eye cancers such as but not limited to ocularmelanoma such as iris melanoma, choroidal melanoma, and cilliary bodymelanoma, and retinoblastoma; vaginal cancers such as squamous cellcarcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamouscell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,and Paget's disease; cervical cancers such as but not limited to,squamous cell carcinoma, and adenocarcinoma; uterine cancers such as butnot limited to endometrial carcinoma and uterine sarcoma; ovariancancers such as but not limited to, ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor; esophageal cancerssuch as but not limited to, squamous cancer, adenocarcinoma, adenoidcyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma,sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell(small cell) carcinoma; stomach cancers such as but not limited to,adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading,diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma, gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, adenocarcinoma,leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers suchas but not limited to squamous cell carcinoma; basal cancers; salivarygland cancers such as but not limited to adenocarcinoma, mucoepidermoidcarcinoma, and adenoidcystic carcinoma; pharynx cancers such as but notlimited to squamous cell cancer, and verrucous; skin cancers such as butnot limited to, basal cell carcinoma, squamous cell carcinoma andmelanoma, superficial spreading melanoma, nodular melanoma, lentigomalignant melanoma, acral lentiginous melanoma; kidney cancers such asbut not limited to renal cell cancer, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America). It is alsocontemplated that cancers caused by aberrations in apoptosis can also betreated by the methods and compositions of the invention. Such cancersmay include, but not be limited to, follicular lymphomas, carcinomaswith p53 mutations, hormone dependent tumors of the breast, prostate andovary, and precancerous lesions such as familial adenomatous polyposis,and myelodysplastic syndromes.

5.5.1.1. Anti-Cancer Therapies

The present invention provides methods of preventing, managing, treatingor ameliorating cancer or one or more symptoms thereof, said methodscomprising administering to a subject in need thereof a liquidformulation of the invention and one or more therapies (e.g.prophylactic or therapeutic agents) other than antibodies (includingantibody fragments thereof) that immunospecifically bind to an IL-9polypeptide. Therapeutic or prophylactic agents include, but are notlimited to, peptides, polypeptides, proteins, fusion proteins, nucleicacid molecules, small molecules, mimetic agents, synthetic drugs,inorganic molecules, and organic molecules. Any agent or therapy (e.g.,chemotherapies, radiation therapies, hormonal therapies, and/orbiological therapies/immunotherapies) which is known to be useful, orwhich has been used or is currently being used for the prevention,treatment, management or amelioration of cancer or one or more symptomsthereof can be used in combination with a liquid formulation of theinvention in accordance with the invention described herein.

In certain embodiments, the anti-cancer agent is an immunomodulatoryagent, such as a chemotherapeutic agent. In certain other embodiments,the anti-cancer agent is an immunomodulatory agent other than achemotherapeutic agent. In other embodiments, the anti-cancer agent isnot an immunomodulatory agent. In specific embodiments, the anti-canceragent is an anti-angiogenic agent. In other embodiments, the anti-canceragent is not an anti-angiogenic agent. In specific embodiments, theanti-cancer agent is an anti-inflammatory agent. In other embodiments,the anti-cancer agent is not an anti-inflammatory agent.

In particular embodiments, the anti-cancer agent is, but not limited to:acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandomate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflomithine hydrochloride; EphA2 inhibitors (e.g.,anti-EphA2 antibodies that result in the phosphorylation of EphA2 andthe degration of EphA2 (see, U.S. Patent Application No. 60/418,213,which is incorporated herein by reference in its entirety);elsamitrucin; enloplatin; enpromate; epipropidine; epirubicinhydrochloride; erbulozole; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide;floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;interleukin II (including recombinant interleukin II, or rIL2),interferon alpha-2a; interferon alpha-2b; interferon alpha-n1;interferon alpha-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; anti-CD2 antibodies (e.g., siplizumab(MedImmune Inc.; International Publication No. WO 02/098370, which isincorporated herein by reference in its entirety)); megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, dioxamycin; diphenyl spiromustine; docetaxel; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; HMG CoAreductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin,lescol, lupitor, lovastatin, rosuvastatin, and simvastatin); hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-I receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP(Biogen, Cambridge, Mass.; International Publication No. WO 93/0686 andU.S. Pat. No. 6,162,432); liarozole; linear polyamine analogue;lipophilic disaccharide peptide; lipophilic platinum compounds;lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinaseinhibitors; menogaril; merbarone; meterelin; methioninase;metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim;mismatched double stranded RNA; mitoguazone; mitolactol; mitomycinanalogues; mitonafide; mitotoxin fibroblast growth factor-saporin;mitoxantrone; mofarotene; molgramostim; monoclonal antibody, humanchorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wallsk; mopidamol; multiple drug resistance gene inhibitor; multiple tumorsuppressor 1-based therapy; mustard anticancer agent; mycaperoxide B;mycobacterial cell wall extract; myriaporone; N-acetyldinaline;N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine;napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronicacid; neutral endopeptidase; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; VITAXIN™ (see U.S. Patent Pub. No. U.S.2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering Integrinα_(v)β3 Antagonists in Combination With Other Prophylactic orTherapeutic Agents”); vorozole; zanoterone; zeniplatin; zilascorb; andzinostatin stimalamer. In another preferred embodiment, an antibodyderivative such as MT103, part of a class of antibody derivatives knownas Bi-Specific T Cell Engagers (BiTE™; MedImmune, Inc.), may also beused in combination with one or more liquid formulations of the presentinvention.

Other examples of anti-cancer agents include, but are not limited to,angiogenesis inhibitors, topoisomerase inhibitors and immunomodulatoryagents (such as chemotherapeutic agents and non-therapeuticimmunomodulatory agents, including but not limited to, anti-T cellreceptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412(Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone andOKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (ProductDesign Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies (e.g.,MEDI-507 (MedImmune, Inc., International Publication Nos. WO 02/098370and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim (Genentech)),and anti-B7 antibodies (e.g., IDEC-114) (IDEC)); anti-cytokine receptorantibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptorantibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptorantibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptorantibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1βantibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8(Abgenix)), and anti-IL-12 antibodies)); CTLA4-immunoglobulin; LFA-3TIP(Biogen, International Publication No. WO 93/08656 and U.S. Pat. No.6,162,432); soluble cytokine receptors (e.g., the extracellular domainof a TNF-α receptor or a fragment thereof, the extracellular domain ofan IL-1β receptor or a fragment thereof, and the extracellular domain ofan IL-6 receptor or a fragment thereof); cytokines or fragments thereof(e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, interferon (IFN)-α, IFN-β,IFN-γ, and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-αantibodies, and anti-IFN-γ antibodies), and antibodies thatimmunospecifically bind to tumor-associated antigens (e.g., HERCEPTIN®).

The invention also encompasses administration of a liquid formulation ofthe invention in combination with radiation therapy comprising the useof x-rays, gamma rays and other sources of radiation to destroy thecancer cells. In preferred embodiments, the radiation treatment isadministered as external beam radiation or teletherapy wherein theradiation is directed from a remote source. In other preferredembodiments, the radiation treatment is administered as internal therapyor brachytherapy wherein a radiaoactive source is placed inside the bodyclose to cancer cells or a tumor mass.

In specific embodiments, patients with breast cancer are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with the administration of aprophylactically or therapeutically effective amount of one or moreother agents useful for breast cancer therapy including but not limitedto: doxorubicin, epirubicin, the combination of doxorubicin andcyclophosphamide (AC), the combination of cyclophosphamide, doxorubicinand 5-fluorouracil (CAF), the combination of cyclophosphamide,epirubicin and 5-fluorouracil (CEF), Herceptin®, tamoxifen, thecombination of tamoxifen and cytotoxic chemotherapy. In certainembodiments, patients with metastatic breast cancer are administered aprophylactically or therapeutically effective amount of one or moreliquid formulations of the invention in combination with theadministration of an effective amount of taxanes such as docetaxel andpaclitaxel. In other embodiments, a prophylactically or therapeuticallyeffective amount of a liquid formulation of the invention isadministered in combination with the administration of aprophylactically or therapeutically effective amount of taxanes plusstandard doxorubicin and cyclophosphamide for adjuvant treatment ofnode-positive, localized breast cancer.

In specific embodiments, patients with prostate cancer are administereda prophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with the administration of aprophylactically or therapeutically effective amount of one or moreother agents useful for prostate cancer therapy including but notlimited to: external-beam radiation therapy, interstitial implantationof radioisotopes (i.e., I¹²⁵, palladium, Iridium), leuprolide or otherLHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide,bicalutamide), steroidal antiandrogens (cyproterone acetate), thecombination of leuprolide and flutamide, estrogens such as DES,chlorotrianisene, ethinyl estradiol, conjugated estrogens U.S.P.,DES-diphosphate, radioisotopes, such as strontium-89, the combination ofexternal-beam radiation therapy and strontium-89, second-line hormonaltherapies such as aminoglutethimide, hydrocortisone, flutamidewithdrawal, progesterone, and ketoconazole, low-dose prednisone, orother chemotherapy regimens reported to produce subjective improvementin symptoms and reduction in PSA level including docetaxel, paclitaxel,estramustine/docetaxel, estramustine/etoposide,estramustine/vinblastine, and estramustine/paclitaxel. In specificembodiments, patients with ovarian cancer are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with a prophylactically ortherapeutically effective amount of one or more other agents useful forovarian cancer therapy including but not limited to: intraperitonealradiation therapy, such as p³² therapy, total abdominal and pelvicradiation therapy, cisplatin, the combination of paclitaxel (Taxol) ordocetaxel (Taxotere) and cisplatin or carboplatin, the combination ofcyclophosphamide and cisplatin, the combination of cyclophosphamide andcarboplatin, the combination of 5-FU and leucovorin, etoposide,liposomal doxorubicin, gemcitabine or topotecan. It is contemplated thata prophylactically or therapeutically effective amount of a liquidformulation of the invention is administered in combination with theadministration Taxol for patients with platinum-refractory disease.Included is the treatment of patients with refractory ovarian cancerincluding administration of: ifosfamide in patients with disease that isplatinum-refractory, hexamethylmelamine (HMM) as salvage chemotherapyafter failure of cisplatin-based combination regimens, and tamoxifen inpatients with detectable levels of cytoplasmic estrogen receptor ontheir tumors. In specific embodiments, patients with bone sarcomas areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with aprophylactically or therapeutically effective amount of one or moreother agents useful for bone sarcoma therapy including but not limitedto: doxorubicin, ifosfamide, cisplatin, high-dose methotrexate,cyclophosphamide, etoposide, vincristine, dactinomycin, and surgery.

In specific embodiments, patients with tumor metastatic to bone areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with aprophylactically or therapeutically effective amount of one or moreother agents useful for bone metastatic tumor therapy including but notlimited to: agents or therapies used in treatment of underlyingmalignancy (non-limiting examples are hormone inhibitors for prostate orbreast cancer metastasized to bone and surgery), radiotherapy(non-limiting examples are strontium 89 and samarium 153, which arebone-seeking radionuclides that can exert antitumor effects and relievesymptoms), and bisphosponates.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (59^(th) ed., 2005).

5.5.2. Proliferative Disorders

The liquid antibody formulations of the invention can be used toprevent, treat, manage, and/or ameliorate a proliferative disorder orone or more symptoms thereof. In a specific embodiment, theproliferative disorder is characterized by aberrant proliferation (e.g.uncontrolled proliferation or lack of proliferation) of cells that IL-9mediates the growth of, including, but not limited to T cells, erythroidprogenitors, B cells, mast cells, eosinophils, neutrophils, and fetalthymocytes.

The present invention provides methods for preventing, treating,managing, and/or ameliorating one or more symptoms of a non-cancerousdisorder (i.e., a disorder that does not have the potential tometasasize) associated with IL-9 mediated cellular hyperproliferation,particularly of epithelial cells (e.g., as in asthma, COPD, lungfibrosis, bronchial hyperresponsiveness, psoriasis, lymphoproliferativedisorder, and seborrheic dermatitis) and endothelial cells (e.g., as inrestenosis, hyperproliferative vascular disease, Behcet's Syndrome,atherosclerosis, and macular degeneration), said methods comprisingadministering to a subject in need thereof an effective amount of one ormore antibodies of the invention. The present invention also providesmethods for preventing, managing, treating, and/or ameliorating anon-cancerous disorder associated with IL-9 mediated cellularhyperproliferation, said methods comprising of administering to asubject in need thereof an effective amount of one or more antibodies ofthe invention and an effective amount of one or more other therapies(e.g., one or more prophylactic or therapeutic agents) other thanantibodies of the invention useful for the prevention, treatment,management, and/or amelioration of said disorder.

The invention provides methods for preventing, treating, managing,and/or ameliorating one or more symptoms of a non-cancerous disorderassociated with IL-9 mediated cellular hyperproliferation in a subjectrefractory to conventional therapies for such disorder, said methodscomprising administering to subject an effective amount of one or moreantibodies, compositions, or combination therapies of the invention. Incertain embodiments, a patient with a non-cancerous disorder associatedwith IL-9 mediated cellular hyperproliferation is refractory to atherapy when the hyperproliferation has not been eradicated and/or thesymptoms have not been alleviated. The determination of whether apatient is refractory can be made either in vivo or in vitro by anymethod known in the art for assaying the effectiveness of a treatment ofnon-cancerous hyperproliferation disorders, using art-accepted meaningsof “refractory” such a context. In various embodiments, a patient with anon-cancerous disorder associated with IL-9 mediated cellularhyperproliferation is refractory when the patient's levels of IL-9remain abnormal and/or if cellular proliferation has not been decreased.The present invention also provides methods for preventing, managing,treating, and/or ameliorating a non-cancerous disorder associated withIL-9 mediated cellular hyperproliferation in a subject refractory toconventional therapies for such disorder, said methods comprising ofadministering to a subject in need thereof an effective amount of one ormore antibody formulations of the invention and an effective amount ofone or more other therapies (e.g., one or more prophylactic ortherapeutic agents) other than antibody formulations of the inventionuseful for the prevention, treatment, management, and/or amelioration ofsaid disorder.

In a specific embodiment, an effective amount of one or more antibodiesof the invention is administered in combination with an effective amountof a liquid formulation of the invention containing an antibody(including antibody fragment thereof), (e.g., 4D4, 4D4H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4) to a subject at risk of or with a proliferative disorder.The liquid antibody formulations of the invention or combinationtherapies of the invention may be used as the first, second, third,fourth, or fifth therapy to prevent, manage, treat, and/or ameliorate aproliferative disorder or one or more symptom thereof. The inventionalso includes methods of preventing, treating, managing, and/orameliorating a proliferative disorder or one or more symptoms thereof ina patient undergoing therapies for other disease or disorders. Theinvention encompasses methods of preventing, managing, treating, and/orameliorating a proliferative disorder or one or more symptoms thereof ina patient before any adverse effects or intolerance to therapies otherthan antibodies of the invention develops. The invention alsoencompasses methods of preventing, managing, treating, and/orameliorating a proliferative disorder or a symptom thereof in patientswho are susceptible to adverse reactions to conventional therapies.

The invention encompasses methods for preventing, treating, managing,and/or ameliorating a proliferative disorder or a symptom thereof in apatient who has proven refractory to therapies other than antibodies,compositions, or combination therapies of the invention. In certainembodiments, a patient with a proliferative disorder is refractory to atherapy when proliferation disorders has not been eradicated and/or thesymptoms have not been alleviated. The determination of whether apatient is refractory can be made either in vivo or in vitro by anymethod known in the art for assaying the effectiveness of a treatment ofproliferative disorders, using art-accepted meanings of “refractory”such a context. In various embodiments, a patient with a proliferativedisorder is refractory when the patient's levels of IL-9 remain abnormaland/or if cellular proliferation has not been decreased.

The present invention provides methods for preventing, treating,managing, and/or ameliorating a proliferative disorder or one or moresymptoms thereof as an alternative to other conventional therapies. Inspecific embodiments, the patient being managed or treated in accordancewith the methods of the invention is to other therapies or issusceptible to adverse reactions from such therapies. The patient may bea person with a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease), aperson with impaired renal or liver function, the elderly, children,infants, persons with neuropsychiatric disorders or those who takepsychotropic drugs, persons with histories of seizures, or persons onmedication that would negatively interact with conventional agents usedto manage or treat a proliferative disorder.

Therapies and dosages, routes of administration, and recommended usageof therapies for preventing, treating, managing, and/or amelioratingproliferative disorders or one or more symptoms thereof are known in theart and have been described in such literature as the Physician's DeskReference (59th ed., 2005).

5.5.3. Inflammatory Disorder Treatment

The liquid formulations of the invention may be administered to asubject in need thereof to prevent, manage, treat or ameliorate aninflammatory disorder (e.g., asthma) or one or more symptoms thereof.The liquid formulations of the invention may also be administered incombination with one or more other therapies, preferably therapiesuseful for the prevention, management, treatment or amelioration of aninflammatory disorder (including, but not limited to the prophylactic ortherapeutic agents listed in Section 5.5.3.1 hereinbelow) to a subjectin need thereof to prevent, manage, treat or ameliorate an inflammatorydisorder or one or more symptoms thereof. In a specific embodiment, theinvention provides a method of preventing, managing, treating orameliorating an inflammatory disorder or one or more symptoms thereof,said method comprising administering to a subject in need thereof a doseof a prophylactically or therapeutically effective amount of a liquidformulation of the invention. In another embodiment, the inventionprovides a method of preventing, managing, treating or ameliorating aninflammatory disorder or one or more symptoms thereof, said methodcomprising administering to a subject in need thereof a dose of aprophylactically or therapeutically effective amount of a liquidformulation of the invention and a dose of a prophylactically ortherapeutically effective amount of one or more therapies (e.g.,prophylactic or therapeutic agents) other than antibodies (includingantibody fragments thereof) that immunospecifically bind to an IL-9polypeptide.

The invention provides methods for managing, treating or amelioratingone or more symptoms of an inflammatory disorder in a subject refractoryto conventional therapies (e.g., methotrexate and a TNF-α antagonist(e.g., REMICADE™ or ENBREL™)) for such an inflammatory disorder, saidmethods comprising administering to said subject a dose of aprophylactically or therapeutically effective amount of a liquidformulation of the invention. The invention also provides methods formanaging, treating or ameliorating one or more symptoms of aninflammatory disorder in a subject refractory to existing single agenttherapies for such an inflammatory disorder, said methods comprisingadministering to said subject a dose of a prophylactically ortherapeutically effective amount of a liquid formulation of theinvention and a dose of a prophylactically or therapeutically effectiveamount of one or more therapies (e.g., prophylactic or therapeuticagents) other than antibodies (including antibody fragments thereof)that immunospecifically bind to an IL-9 polypeptide. The invention alsoprovides methods for managing or treating an inflammatory disorder byadministering a liquid formulation of the invention in combination withany other treatment to patients who have proven refractory to othertreatments but are no longer on these treatments. The invention alsoprovides alternative methods for the treatment of an inflammatorydisorder where another therapy has proven or may prove too toxic, i.e.,results in unacceptable or unbearable side effects, for the subjectbeing treated. For example, the liquid formulations of the invention maybe administered to a subject, wherein the subject is refractory to a TNFantagonist or methotrexate. Further, the invention provides methods forpreventing the recurrence of an inflammatory disorder in patients thathave been treated and have no disease activity by administering a liquidformulation of the invention.

Inflammatory disorders that can be treated by the methods encompassed bythe invention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, osteoarthritis, spondyloarthropathies (e.g., psoriaticarthritis, ankylosing spondylitis, Reiter's Syndrome (reactivearthritis), inflammatory osteolysis, Wilson's disease and chronicinflammation resulting from chronic viral or bacteria infections. Asdescribed herein in Section 5.5.4.1, some autoimmune disorders areassociated with an inflammatory condition.

Anti-inflammatory therapies and their dosages, routes of administrationand recommended usage are known in the art and have been described insuch literature as the Physician's Desk Reference (59th ed., 2005).

5.5.3.1. Anti-Inflammatory Therapies

The present invention provides methods of preventing, managing, treatingor ameliorating an inflammatory disorder or one or more symptomsthereof, said methods comprising administering to a subject in needthereof a liquid formulation of the invention and one or more therapies(e.g., prophylactic or therapeutic agents other than antibodies(including antibody fragments thereof) that immunospecifically bind toan IL-9 polypeptide. Any agent or therapy which is known to be useful,or which has been used or is currently being used for the prevention,management, treatment or amelioration of an inflammatory disorder or oneor more symptoms thereof can be used in combination with a liquidformulation of the invention in accordance with the invention describedherein.

Any anti-inflammatory agent, including agents useful in therapies forinflammatory disorders, well-known to one of skill in the art can beused in the compositions and methods of the invention. Non-limitingexamples of anti-inflammatory agents include non-steroidalanti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs,anticholinergics (e.g., atropine sulfate, atropine methylnitrate, andipratropium bromide (ATROVENT™)), beta2-agonists (e.g., abuterol(VENTOLIN™ and PROVENTIL™), bitolterol (TORNALATE™), levalbuterol(XOPONEX™), metaproterenol (ALUPENT™), pirbuterol (MAXAIR™), terbutlaine(BRETHAIRE™ and BRETHINE™), albuterol (PROVENTIL™, REPETABS™, andVOLMAX™), formoterol (FORADIL AEROLIZER™), and salmeterol (SEREVEN™ andSEREVENT DISKUS™)), and methylxanthines (e.g., theophylline (UNIPHYL™,THEO-DUR™, SLO-BID™, AND TEHO-42™)). Examples of NSAIDs include, but arenot limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac(VOLTAREN™), etodolac (LODINE™), fenoprofen (NALFON™), indomethacin(INDOCIN™), ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone(RELAFEN™), sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib(VIOXX™), naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) andnabumetone (RELAFEN™). Such NSAIDs function by inhibiting acyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examples of steroidalanti-inflammatory drugs include, but are not limited to,glucocorticoids, dexamethasone (DECADRON™), corticosteroids (e.g.,methylprednisolone (MEDROL™)), cortisone, hydrocortisone, prednisone(PREDNISONE™ and DELTASONE™), prednisolone (PRELONE™ and PEDIAPRED™),triamcinolone, azulfidine, and inhibitors of eicosanoids (e.g.,prostaglandins, thromboxanes, and leukotrienes (see Table 2, infra, fornon-limiting examples of leukotriene and typical dosages of suchagents)).

In a specific embodiment, an effective amount of one or more antibodiesof the invention is administered in combination with an effective amountof VITAXIN™ (MedImmune, Inc., International Publication No. WO 00/78815,International Publication No. WO 02/070007 A1, dated Sep. 12, 2002,entitled “Methods of Preventing or Treating Inflammatory or AutoimmuneDisorders by Administering Integrin AlphaV Beta3 Antagonists,”International Publication No. WO 03/075957 A1, dated Sep. 18, 2003,entitled “The Prevention or Treatment of Cancer Using IntegrinAlphaVBeta3 Antagonists in Combination With Other Agents,” U.S. PatentPub. No. U.S. 2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods ofPreventing or Treating Inflammatory or Autoimmune Disorders byAdministering Integrin α_(v)β3 Antagonists in Combination With OtherProphylactic or Therapeutic Agents,” and International Publication No.WO 03/075741 A2, dated Sep. 18, 2003, entitled, “Methods of Preventingor Treating Disorders by Administering an Integrin αvβ₃ Antagonist inCombination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate,”each of which is incorporated herewith by reference in its entirety) toa subject to prevent, treat, manage, and/or ameliorate an inflammatorydisorder or one or more symptoms thereof. In another embodiment, aneffective amount of one or more antibodies of the invention isadministered in combination with an effective amount of siplizumab(MedImmune, Inc., International Publication No. WO 02/069904) to asubject to prevent, treat, manage, and/or ameliorate an inflammatorydisorder or one or more symptoms thereof. In another embodiment, aneffective amount of one or more antibodies of the invention isadministered in combination with an effective amount of one or moreEphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (MedImmune,Inc.; International Publication No. WO 02/102974 A4, dated Dec. 27,2002, entitled “Mutant Proteins, High Potency Inhibitory Antibodies andFIMCH Crystal Structure,” International Publication No. 03/094859 A2,dated Nov. 20, 2003, entitled “EphA2 Monoclonal Antibodies and Methodsof Use Thereof,” U.S. Appn. No. 10/436,783; and U.S. Appn. No.60/379,368, each of which is incorporated herewith by reference)) to asubject to prevent, treat, manage, and/or ameliorate an inflammatorydisorder or one or more symptoms thereof. In yet another preferredembodiment, an effective amount of one or more antibodies of theinvention is administered in combination with an effective amount ofVITAXIN™, siplizumab, and/or EphA2 inhibitor to a subject to prevent,treat, manage, and/or ameliorate an inflammatory disorder or one or moresymptoms thereof.

In one embodiment, an effective amount of one or more antibodyformulations of the invention is administered in combination with a mastcell protease inhibitor to a subject at risk of or with an inflammatorydisorder. In another embodiment, the mast cell protease inhibitor is atryptase kinase inhibitor, such as, but not limited to GW-45, GW-58, andgenisteine. In a specific embodiment, the mast cell protease inhibitoris phosphatidylinositide-3′ (PI3)-kinase inhibitors, such as, but notlimited to calphostin C. In another embodiment, the mast cell proteaseinhibitor is a protein kinase inhibitor such as, but not limited tostaurosporine. In accordance with this embodiments, the mast cellprotease inhibitor is preferably administered locally to the affectedarea.

Specific examples of immunomodulatory agents which can be administeredin combination with a liquid formulation of the invention to a subjectwith an inflammatory disorder include, but are not limited to,methothrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide),anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g.,cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthocloneand OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion(Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)),anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate),anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies(e.g., MEDI-507 (Medlmmune, Inc., International Publication Nos. WO02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC));anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies,anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)),anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-1βantibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8(Abgenix)), and anti-IL-12 antibodies)); CTLA4-immunoglobulin; LFA-3TIP(Biogen, International Publication No. WO 93/08656 and U.S. Pat. No.6,162,432); soluble cytokine receptors (e.g., the extracellular domainof a TNF-a receptor or a fragment thereof, the extracellular domain ofan IL-1β receptor or a fragment thereof, and the extracellular domain ofan IL-6 receptor or a fragment thereof); cytokines or fragments thereof(e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, interferon (IFN)-α, IFN-β,IFN-γ, and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-αantibodies, and anti-IFN-γ antibodies).

Any TNF-α antagonist well-known to one of skill in the art can be usedin the compositions and methods of the invention. Non-limiting examplesof TNF-α antagonists which can be administered in combination with aliquid formulation of the invention to a subject with an inflammatorydisorder include proteins, polypeptides, peptides, fusion proteins,antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal,Fvs, ScFvs, Fab fragments, F(ab)₂ fragments, and antigen-bindingfragments thereof) such as antibodies that immunospecifically bind toTNF-α, nucleic acid molecules (e.g., antisense molecules or triplehelices), organic molecules, inorganic molecules, and small moleculesthat blocks, reduces, inhibits or neutralizes the function, activityand/or expression of TNF-α. In various embodiments, a TNF-α antagonistreduces the function, activity and/or expression of TNF-α by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or at least 99% relative to a controlsuch as phosphate buffered saline (PBS). Examples of antibodies thatimmunospecifically bind to TNF-α include, but are not limited to,infliximab (REMICADE™; Centacor), D2E7 (Abbott Laboratories/KnollPharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known asHUMICADE™ and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), andTN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91:2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA89:7375-7379). The present invention also encompasses the use ofantibodies that immunospecifically bind to TNF-α disclosed in thefollowing U.S. Pat. Nos. in the compositions and methods of theinvention: 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380;5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272;5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452;5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and6,171,787; each of which are herein incorporated by reference in theirentirety. Examples of soluble TNF-α receptors include, but are notlimited to, sTNF-R1 (Amgen), etanercept (ENBREL™; Immunex) and its rathomolog RENBREL™, soluble inhibitors of TNF-α derived from TNFrI, TNFrII(Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335), and TNF-αInh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192).

Other TNF-α antagonists encompassed by the invention include, but arenot limited to, IL-10, which is known to block TNF-α production viainterferon γ-activated macrophages (Oswald et al. 1992, Proc. Natl.Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc.Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-1(Serono/Yeda), the vaccine CytoTAb (Protherics), antisense molecule104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide (Celgene),CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207(AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469(Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim(Dynavax), auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals),quinacrine (mepacrine dichlorohydrate), tenidap (Enablex), Melanin(Large Scale Biological), and anti-p38 MAPK agents by Uriach.

Non-limiting examples of anti-inflammatory agents which can beadministered in combination with a liquid formulation of the inventionto a subject with an inflammatory disorder include non-steroidalanti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs,beta-agonists, anticholingeric agents, and methyl xanthines. Examples ofNSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib(CELEBREX™), diclofenac (VOLTAREN™), etodolac (LODINE™), fenoprofen(NALFON™), indomethacin (INDOCIN™), ketoralac (TORADOL™), oxaprozin(DAYPRO™), nabumentone (RELAFEN™), sulindac (CLINORIL™), tolmentin(TOLECTIN™), rofecoxib (VIOXX™), naproxen (ALEVE™, NAPROSYN™),ketoprofen (ACTRON™) and nabumetone (RELAFEN™). Such NSAIDs function byinhibiting a cyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examplesof steroidal anti-inflammatory drugs include, but are not limited to,glucocorticoids, dexamethasone (DECADRON™), cortisone, hydrocortisone,prednisone (DELTASONE™), prednisolone, triamcinolone, azulfidine, andeicosanoids such as prostaglandins, thromboxanes, and leukotrienes.

In specific embodiments, patients with osteoarthritis are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with other agents ortherapies useful for osteoarthritis prevention, treatment, management oramelioration including but not limited to: analgesics (non-limitingexamples are acetaminophen, in a dose up to 4000 mg/d; phenacetin; andtramadol, in a daily dose in the range of 200 to 300 mg); NSAIDs(non-limiting examples include but not limited to, aspirin, diflunisal,diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin,oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin. Low doseNSAIDs are preferred, e.g., ibuprofen at 1200 mg/d, naproxen at 500mg/d. A gastroprotective agent, e.g., misoprostol, famotidine oromeprazole, is preferred to use concurrently with a NSAID);nonacetylated salicylates including but not limited to salsalate;cyclooxygenase (Cox)-2-specific inhibitors (CSIs), including but notlimited to, celecoxib and rofecoxib; intra- or periarticular injectionof a depot glucocorticoid preparation; intra-articular injection ofhyaluronic acid; capsaicin cream; copious irrigation of theosteroarthritis knee to flush out fibrin, cartilage shards and otherdebris; and joint replacement surgery. The liquid formulations of theinvention can also be used in combination with other nonpharmacologicmeasures in prevention, treatment, management and amelioration ofosteoarthritis including but not limited to: reduction of joint loading(non-limiting examples are correction of poor posture, support forexcessive lumbar lordosis, avoid excessive loading of the involvedjoint, avoid prolonged standing, kneeling and squatting); application ofheat to the affected joint; aerobic exercise and other physicaltherapies.

In specific embodiments, patients with rheumatoid arthritis areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with other agents ortherapies useful in prevention, treatment, management and ameliorationof rheumatoid arthritis including but not limited to: NSAIDs(non-limiting examples include but not limited to, aspirin, diflunisal,diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin,oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.);analgesics (non-limiting examples are acetaminophen, phenacetin andtramadol); CSIs including but not limited to, celecoxib and rofecoxib;glucocorticoids (preferably low-dose oral glucocorticoids, e.g., <7.5mg/d prednisone, or monthly pulses with high-dose glucocorticoids, orintraarticular glucocorticoids); disease-modifying antirheumatic drugs(DMARDs) including but not limited to, methotrexate (preferably givenintermittent low dose, e.g., 7.5-30 mg once weekly), gold compounds(e.g., gold salts), D-penicillamine, the antimalarials (e.g.,chloroquine), and sulfasalazine; TNF-α neutralizing agents including butnot limited to, etanercept and infliximab; immunosuppressive andcytotoxic agents (examples include but not limited to, azathioprine,leflunomide, cyclosporine, and cyclophosphamide), and surgery (examplesinclude but not limited to, arthroplasties, total joint replacement,reconstructive hand surgery, open or arthroscopic synovectomy, and earlytenosynovectomy of the wrist). The liquid formulations of the inventionmay also be used in combination with other measures in prevention,treatment, management and amelioration of the rheumatoid arthritisincluding but not limited to: rest, splinting to reduce unwanted motionof inflamed joint, exercise, used of a variety of orthotic and assistivedevices, and other physical therapies. The liquid formulations of theinvention may also be used in combination with some nontraditionalapproaches in prevention, treatment, management and amelioration ofrheumatoid arthritis including but not limited to, diets (e.g.,substituting omega-3 fatty acids such as eicosapentaenoic acid found incertain fish oils for dietary omega-6 essential fatty acids found inmeat), vaccines, hormones and topical preparations.

In specific embodiments, patients with chronic obstructive pulmonarydisease (COPD) are administered a prophylactically or therapeuticallyeffective amount of a liquid formulation of the invention in combinationwith other agents or therapies useful in prevention, treatment,management and amelioration of COPD including but not limited to:bronchodilators including but not limited to, short- and long-actingβ₂-adrenergic agonists (examples of short-acting β₂ agonist include butnot limited to, albuterol, pirbuterol, terbutaline, and metaproterenol;examples of long-acting β₂ agonist include but not limited to, oralsustained-release albuterol and inhaled salmeterol), anticholinergics(examples include but not limited to ipratropium bromide), andtheophylline and its derivatives (therapeutic range for theophylline ispreferably 10-20 μg/mL); glucocorticoids; exogenous α₁AT (e.g., α₁ATderived from pooled human plasma administered intravenously in a weeklydose of 60 mg/kg); oxygen; lung transplantation; lung volume reductionsurgery; endotracheal intubation, ventilation support; yearly influenzavaccine and pneumococcal vaccination with 23-valent polysaccharide;exercise; and smoking cessation.

In specific embodiments, patients with pulmonary fibrosis areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with an effectiveamount of one or more other agents useful for pulmonary fibrosis therapyincluding but not limited to: oxygen; corticosteroids (a non-limitingexample is to administer daily prednisone beginning at 1-1.5 mg/kg/d (upto 100 mg/d) for six weeks and tapering slowly over 3-6 months to aminimum maintenance dose of 0.25 mg/kg/d); cytotoxic drugs (non-limitingexamples are cyclophosphamide at 100-120 mg orally once daily, andazathioprine at 3 mg/kg up to 200 mg orally once daily); bronchodilators(non-limiting examples are short- and long-acting β₂-adrenergicagonists, anticholinergics, and theophylline and its derivatives); andantihistamines (non-limiting examples are diphenhydramine anddoxylamine).

In specific embodiments, patients with asthma are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with an effective amount ofone or more other agents useful for asthma therapy. Non-limitingexamples of such agents include adrenergic stimulants (e.g.,catecholamines (e.g., epinephrine, isoproterenol, and isoetharine),resorcinols (e.g., metaproterenol, terbutaline, and fenoterol), andsaligenins (e.g., salbutamol)), adrenocorticoids, blucocorticoids,corticosteroids (e.g., beclomethadonse, budesonide, flunisolide,fluticasone, triamcinolone, methylprednisolone, prednisolone, andprednisone), other steroids, beta2-agonists (e.g., albtuerol,bitolterol, fenoterol, isoetharine, metaproterenol, pirbuterol,salbutamol, terbutaline, formoterol, salmeterol, and albutamolterbutaline), anti-cholinergics (e.g., ipratropium bromide andoxitropium bromide), IL-4 antagonists (including antibodies), IL-5antagonists (including antibodies), IL-13 antagonists (includingantibodies), PDE4-inhibitor, NF-Kappa-β inhibitor, VLA-4 inhibitor, CpG,anti-CD23, selectin antagonists (TBC 1269), mast cell proteaseinhibitors (e.g., tryptase kinase inhibitors (e.g., GW-45, GW-58, andgenisteine), phosphatidylinositide-3′ (PI3)-kinase inhibitors (e.g.,calphostin C), and other kinase inhibitors (e.g., staurosporine) (seeTemkin et al., 2002 J Immunol 169(5):2662-2669; Vosseller et al., 1997Mol. Biol. Cell 8(5):909-922; and Nagai et al., 1995 Biochem Biophys ResCommun 208(2):576-581)), a C3 receptor antagonists (includingantibodies), immunosuppressant agents (e.g., methotrexate and goldsalts), mast cell modulators (e.g., cromolyn sodium (INTAL™) andnedocromil sodium (TILADE™)), and mucolytic agents (e.g.,acetylcysteine)). In a specific embodiment, the anti-inflammatory agentis a leukotriene inhibitor (e.g., montelukast (SINGULAIR™), zafirlukast(ACCOLATE™), pranlukast (ONON™), or zileuton (ZYFLO™) (see Table 2)).

TABLE 2 Leukotriene Inhibitors for Asthma Therapy Leukotriene ModifierUsual Daily Dosage Montelukast (SINGULAIR ™) 4 mg for 2-5 years old 5 mgfor 6 to 15 years old 10 mg for 15 years and older Zafirlukast(ACCOLATE ™) 10 mg b.i.d. for 5 to 12 years old twice daily 20 mg b.i.d.for 12 years or older twice daily Pranlukast (ONON ™) Only avialable inAsia Zyleuton (ZYFLO ™) 600 mg four times a day for 12 years and older

In specific embodiments, patients with allergy are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with an effective amount ofone or more other agents useful for allergy therapy. Non-limitingexamples of such agents include antimediator drugs (e.g., antihistamine,see Table 3, infra for non-limiting examples of antihistamine andtypical dosages of such agents), corticosteroids, decongestants,sympathomimetic drugs (e.g., α-adrenergic and β-adrenergic drugs),TNX901 (Leung et al., 2003, N Engl J Med 348(11):986-993), IgEantagonists (e.g., antibodies rhuMAb-E25 omalizumab (see Finn et al.,2003 J Allergy Clin Immuno 111(2):278-284; Corren et al., 2003 J AllergyClin Immuno 111(1):87-90; Busse and Neaville, 2001 Curr Opin AllergyClin Immuno 1(1):105-108; and Tang and Powell, 2001, Eur J Pediatr160(12): 696-704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int ArchAllergy Immuno 128(1):24-32), and mAB Hu-901 (see van Neerven et al.,2001 Int Arch Allergy Immuno 124(1-3):400), theophylline and itsderivatives, glucocorticoids, and immunotherapies (e.g., repeatedlong-term injection of allergen, short course desensitization, and venomimmunotherapy).

TABLE 3 H₁ Antihistamines Chemical class and representative drugs Usualdaily dosage Ethanolamine Diphehydramine 25-50 mg every 4-6 hoursClemastine 0.34-2.68 mg every 12 hours Ethylenediamine Tripelennamine25-50 mg every 4-6 hours Alkylamine Brompheniramine 4 mg every 4-6hours; or 8-12 mg of SR form every 8-12 hour Chlorpheniramine 4 mg every4-6 hours; or 8-12 mg of SR form every 8-12 hour Triprolidine (1.25 mg/5ml) 2.5 mg every 4-6 hours Phenothiazine Promethazine 25 mg at bedtimePiperazine Hydroxyzine 25 mg every 6-8 hours Piperidines Astemizole(nonsedating) 10 mg/d Azatadine 1-2 mg every 12 hours Cetirzine 10 mg/dCyproheptadine 4 mg every 6-8 hour Fexofenadine (nonsedating) 60 mgevery 12 hours Loratidine (nonsedating) 10 mg every 24 hours

5.5.4. Autoimmune Disorder Treatment

The liquid formulations of the invention may be administered to asubject in need thereof to prevent, manage, treat or ameliorate anautoimmune disorder or one or more symptoms thereof. The liquidformulations of the invention may also be administered in combinationwith one or more other therapies, preferably therapies useful for theprevention, management or treatment of an autoimmune disorder(including, but not limited to the prophylactic or therapeutic agentslisted in Section 5.5.4.1 hereinbelow) to a subject in need thereof toprevent, manage, treat or ameliorate an autoimmune disorder or one ormore symptoms thereof. In a specific embodiment, the invention providesa method of preventing, managing, treating or ameliorating an autoimmunedisorder or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof a dose of a prophylacticallyor therapeutically effective amount of a liquid formulation of theinvention. In another embodiment, the invention provides a method ofpreventing, managing, treating or ameliorating an autoimmune disorder orone or more symptoms thereof, said method comprising administering to asubject in need thereof a dose of a prophylactically or therapeuticallyeffective amount of a liquid formulation of the invention and a dose ofa prophylactically or therapeutically effective amount of one or moretherapies (e.g., prophylactic or therapeutic agents) other thanantibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide.

The invention provides methods for managing, treating or ameliorating anautoimmune disorder or one or more symptoms thereof in a subjectrefractory to conventional therapies for such an autoimmune disorder,said methods comprising administering to said subject a dose of aprophylactically or therapeutically effective amount of a liquidformulation of the invention. The invention also provides methods formanaging, treating or ameliorating an autoimmune disorder or one or moresymptoms thereof in a subject refractory to existing single agenttherapies for such an autoimmune disorder, said methods comprisingadministering to said subject a dose of a prophylactically ortherapeutically effective amount of a liquid formulation of theinvention and a dose of a prophylactically or therapeutically effectiveamount of one or more therapies (e.g., prophylactic or therapeuticagents) other than antibodies (including antibody fragments thereof)that immunospecifically bind to an IL-9 polypeptide. The invention alsoprovides methods for managing, treating or ameliorating an autoimmunedisorder or one or more symptoms thereof by administering a liquidformulation of the invention in combination with any other treatment topatients who have proven refractory to other treatments but are nolonger on these treatments. The invention also provides alternativemethods for the management or treatment of an autoimmune disorder whereanother therapy has proven or may prove too toxic, i.e., results inunacceptable or unbearable side effects, for the subject being treated.Particularly, the invention provides alternative methods for themanagement or treatment of an autoimmune disorder where the patient isrefractory to other therapies. Further, the invention provides methodsfor preventing the recurrence of an autoimmune disorder in patients thathave been treated and have no disease activity by administering a liquidformulation of the invention.

In autoimmune disorders, the immune system triggers an immune responsewhen there are no foreign substances to fight and the body's normallyprotective immune system causes damage to its own tissues by mistakenlyattacking self. There are many different autoimmune disorders whichaffect the body in different ways. For example, the brain is affected inindividuals with multiple sclerosis, the gut is affected in individualswith Crohn's disease, and the synovium, bone and cartilage of variousjoints are affected in individuals with rheumatoid arthritis. Asautoimmune disorders progress destruction of one or more types of bodytissues, abnormal growth of an organ, or changes in organ function mayresult. The autoimmune disorder may affect only one organ or tissue typeor may affect multiple organs and tissues. Organs and tissues commonlyaffected by autoimmune disorders include red blood cells, blood vessels,connective tissues, endocrine glands (e.g., the thyroid or pancreas),muscles, joints, and skin. Examples of autoimmune disorders that can betreated by the methods of the invention include, but are not limited to,alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Ménière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis.

Autoimmune therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (59th ed., 2005).

5.5.4.1. Autoimmune Disorder Therapies

The present invention provides methods of preventing, managing, treatingor ameliorating an autoimmune disorder or one or more symptoms thereof,said methods comprising administering to a subject in need thereof aliquid formulation of the invention and one or more therapies (e.g.,prophylactic or therapeutic agents) other than antibodies (includingantibody fragments thereof) that immunospecifically bind to an IL-9polypeptide. Any agent or therapy which is known to be useful, or whichhas been used or is currently being used for the prevention, management,treatment or amelioration of an autoimmune disorder or one or moresymptoms thereof can be used in combination with a liquid formulation ofthe invention in accordance with the invention described herein.Examples of such agents include, but are not limited to,immunomodulatory agents, anti-inflammatory agents and TNF-α antagonists.Specific examples of immunomodulatory agents, anti-inflammatory agentsand TNF-α antagonists which can be used in combination with a liquidformulation of the invention for the prevention, management, treatmentor amelioration of an autoimmune disorder are disclosed herein above.

In specific embodiments, patients with multiple sclerosis (MS) areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with other agents ortherapies useful in prevention, treatment, management and ameliorationof MS including but not limited to: IFN-β1b (Betaseron) (e.g., 8.0million international unites (MIU) is administered by subcutaneousinjection every other day); IFN-β1a (Avonex) (e.g., 6.0 MIU isadministered by intramuscular injection once every week); glatirameracetate (Copaxone) (e.g., 20 mg is administered by subcutaneousinjection every day); mitoxantrone (e.g., 12 mg/m² is administered byintravenous infusion every third month); azathioprine (e.g., 2-3 mg/kgbody weight is administered orally each day); methotrexate (e.g., 7.5 mgis administered orally once each week); cyclophosphamide; intravenousimmunoglobulin (e.g., 0.15-0.2 g/kg body weight administered monthly forup to 2 years); glucocorticoids; methylprednisolone (e.g., administeredin bimonthly cycles at high doses); 2-chlorodeoxyadenosine (cladribine);baclofen (e.g., 15 to 80 mg/d in divided doses, or orally in higherdoses up to 240 mg/d, or intrathecally via an indwelling catheter);cycloenzaprine hydrochloride (e.g., 5-10 mg bid or tid); clonazepam(e.g., 0.5 to 1.0 mg tid, including bedtime dose); clonidinehydrochloride (e.g., 0.1 to 0.2 mg tid, including a bedtime dose);carbamazepine (e.g., 100-1200 mg/d in divided, escalating doses);gabapentin (e.g., 300-3600 mg/d); dilantin (e.g., 300-400 mg/d);amitriptyline (e.g., 25-150 mg/d); baclofen (e.g., 10-80 mg/d);primidone (e.g., 125-250 mg bid or tid); ondansetron (e.g., 4 to 8 mgbid or tid); isoniazid (e.g., up to 1200 mg in divided doses);oxybutynin (e.g., 5 mg bid or tid); tolterodine (e.g., 1-2 mg bid);propantheline (e.g., 7.5 to 15 mg qid); bethanecol (e.g., 10-50 mg tidor qid); terazosin hydrochloride (e.g., 1-5 mg at bedtime); sildenafilcitrate (e.g., 50-100 mg po prn); amantading (e.g., 100 mg bid);pemoline (e.g., 37.5 mg bid); high dose vitamins; calcium orotate;gancyclovir; antibiotic; and plasma exchange.

In specific embodiments, patients with psoriasis are administered aprophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with other agents ortherapies useful in prevention, treatment, management and ameliorationof psoriasis including but not limited to: topical steroid cream orointment; tar (examples including but not limited to, Estar, Psorigel,Fototar cream, and LCD 10% in Nutraderm lotion or mixed directly withtriamcinolone 0.1% cream); occlusion; topical vitamin D analogue (anon-limiting example is calcipotriene ointment); ultraviolet light; PUVA(psoralen plus ultraviolet A); methotrexate (e.g., up to 25 mg onceweekly or in divided doses every 12 hours for three doses once a week);synthetic retinoid (a non-limiting examples is etretinate, e.g., indosage of 0.5-1 mg/kg/d); immunomodulatory therapy (a non-limitingexample is cyclosporine); sulfasalazine (e.g., in dosages of 1 g threetimes daily).

In specific embodiments, patients with Crohn's disease are administereda prophylactically or therapeutically effective amount of a liquidformulation of the invention in combination with other agents ortherapies useful in prevention, treatment, management and ameliorationof Crohn's disease including but not limited to: antidiarrheals (e.g.,loperamide 2-4 mg up to 4 times a day, diphenoxylate with atropine 1tablet up to 4 times a day, tincture of opium 8-15 drops up to 4 times aday, cholestyramine 2-4 g or colestipol 5 g once or twice daily),antispasmodics (e.g., propantheline 15 mg, dicyclomine 10-20 mg, orhyoscyamine 0.125 mg given before meals), 5-aminosalicylic acid agents(e.g., sulfasalazine 1.5-2 g twice daily, mesalamine (ASACOL®) and itsslow release form (PENTASA®), especially at high dosages, e.g., PENTASA®1 g four times daily and ASACOL® 0.8-1.2 g four times daily),corticosteroids, immunomodulatory drugs (e.g., azathioprine (1-2 mg/kg),mercaptopurine (50-100 mg), cyclosporine, and methotrexate),antibiotics, TNF inhibitors (e.g., inflixmab (REMICADE®)),immunosuppressive agents (e.g., tacrolimus, mycophenolate mofetil, andthalidomide), anti-inflammatory cytokines (e.g., IL-10 and IL-11),nutritional therapies, enteral therapy with elemental diets (e.g.,Vivonex for 4 weeks), and total parenteral nutrition.

In specific embodiments, patients with lupus erythematosus areadministered a prophylactically or therapeutically effective amount of aliquid formulation of the invention in combination with other agents ortherapies useful in prevention, treatment, management and ameliorationof lupus erythematosus including but not limited to: antimalarials(including but not limited to, hydroxychloroquine); glucocorticoids(e.g., low dose, high dose, or high-dose intravenous pulse therapy canbe used); immunosuppressive agents (including but not limited to,cyclophosphamide, chlorambucil, and azanthioprine); cytotoxic agents(including but not limited to methotrexate and mycophenolate mofetil);androgenic steroids (including but not limited to danazol); andanticoagulants (including but not limited to warfarin).

In a specific embodiment, an prophylactically or therapeuticallyeffective amount of one or more liquid antibody formulations of theinvention is administered in combination with an effective amount ofVITAXIN™ (MedImmune, Inc., International Publication No. WO 00/78815,International Publication No. WO 02/070007 A1, dated Sep. 12, 2002,entitled “Methods of Preventing or Treating Inflammatory or AutoimmuneDisorders by Administering Integrin AlphaV Beta3 Antagonists,”International Publication No. WO 03/075957 A1, dated Sep. 18, 2003,entitled “The Prevention or Treatment of Cancer Using IntegrinAlphaVBeta3 Antagonists in Combination With Other Agents,” U.S. PatentPub. No. U.S. 2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods ofPreventing or Treating Inflammatory or Autoimmune Disorders byAdministering Integrin α_(v)β3 Antagonists in Combination With OtherProphylactic or Therapeutic Agents,” and International Publication No.WO 03/075741 A2, dated Sep. 18, 2003, entitled, “Methods of Preventingor Treating Disorders by Administering an Integrin αvβ₃ Antagonist inCombination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate,”each of which is incorporated herewith by reference in its entirety) toa subject to prevent, treat, manage, and/or ameliorate an autoimmunedisorder or one or more symptoms thereof. In another preferredembodiment, an effective amount of one or more antibodies of theinvention is administered in combination with an effective amount ofsiplizumab (MedImmune, Inc., International Publication No. WO 02/069904)to a subject to prevent, treat, manage, and/or ameliorate an autoimmunedisorder or one or more symptoms thereof. In another embodiment, aneffective amount of one or more antibodies of the invention isadministered in combination with an effective amount of one or moreEphA2 inhibitors (e.g., one or more anti-EphA2 antibodies (MedImmune,Inc.; International Publication No. WO 02/102974 A4, dated Dec. 27,2002, entitled “Mutant Proteins, High Potency Inhibitory Antibodies andFIMCH Crystal Structure,” International Publication No. 03/094859 A2,dated Nov. 20, 2003, entitled “EphA2 Monoclonal Antibodies and Methodsof Use Thereof,” U.S. application Ser. No. 10/436,783; and U.S. Appn.No. 60/379,368, each of which is incorporated herewith by reference)) toa subject to prevent, treat, manage, and/or ameliorate an autoimmunedisorder or one or more symptoms thereof. In yet another embodiment, aneffective amount of one or more antibodies of the invention isadministered in combination with an effective amount of VITAXIN™,siplizumab, and/or EphA2 inhibitor to a subject to prevent, treat,manage, and/or ameliorate an autoimmune disorder or one or more symptomsthereof.

The antibody formulations of the invention or combination therapies ofthe invention may be used as the first, second, third, fourth, or fifththerapy to prevent, manage, treat, and/or ameliorate an autoimmunedisorder or one or more symptom thereof. The invention also includesmethods of preventing, treating, managing, and/or ameliorating anautoimmune disorder or one or more symptoms thereof in a patientundergoing therapies for other disease or disorders. The inventionencompasses methods of preventing, managing, treating, and/orameliorating an autoimmune disorder or one or more symptoms thereof in apatient before any adverse effects or intolerance to therapies otherthan antibodies of the invention develops. The invention alsoencompasses methods of preventing, treating, managing, and/orameliorating an autoimmune disorder or a symptom thereof in refractorypatients. The invention encompasses methods for preventing, treating,managing, and/or ameliorating a proliferative disorder or a symptomthereof in a patient who has proven refractory to therapies other thanantibodies, compositions, or combination therapies of the invention. Thedetermination of whether a patient is refractory can be made either invivo or in vitro by any method known in the art for assaying theeffectiveness of a treatment of autoimmune disorders, using art-acceptedmeanings of “refractory” such a context. In certain embodiments, apatent with an autoimmune disorder is refractory to a therapy when oneor more symptoms of an autoimmune disorder is not prevented, managed,and/or alleviated. The invention also encompasses methods of preventing,managing, treating, and/or ameliorating an autoimmune disorder or asymptom thereof in patients who are susceptible to adverse reactions toconventional therapies.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating an autoimmune disorder or one or moresymptoms thereof as an alternative to other conventional therapies. Inspecific embodiments, the patient being managed or treated in accordancewith the methods of the invention is refractory to other therapies or issusceptible to adverse reactions from such therapies. The patient may bea person with a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease,patients with broncho-pulmonary dysplasia, patients with congenitalheart disease, patients with cystic fibrosis, patients with acquired orcongenital heart disease, and patients suffering from an infection), aperson with impaired renal or liver function, the elderly, children,infants, infants born prematurely, persons with neuropsychiatricdisorders or those who take psychotropic drugs, persons with historiesof seizures, or persons on medication that would negatively interactwith conventional agents used to prevent, manage, treat, or ameliorate aviral respiratory infection or one or more symptoms thereof.

Autoimmune therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (59th ed., 2005).

5.5.5. Viral Infections

One or more antibody formulations of the invention can be administeredto a subject to prevent, treat, manage, and/or ameliorate a viralinfection or one or more symptoms thereof. One or more antibodyformulations of the invention may be administered in combination withone or more other therapies (e.g., one or more prophylactic ortherapeutic agents) other than antibody formulations of the inventionuseful for the prevention, treatment, management, or amelioration of aviral infection to a subject predisposed to or with a viral infection,preferably a respiratory viral infection. Non-limiting examples ofanti-viral agents include proteins, polypeptides, peptides, fusionproteins antibodies, nucleic acid molecules, organic molecules,inorganic molecules, and small molecules that inhibit and/or reduce theattachment of a virus to its receptor, the internalization of a virusinto a cell, the replication of a virus, or release of virus from acell. In particular, anti-viral agents include, but are not limited to,nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir,vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet,amantadine, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, and AZT.

In specific embodiments, the anti-viral agent is an immunomodulatoryagent that is immunospecific for a viral antigen. As used herein, theterm “viral antigen” includes, but is not limited to, any viral peptide,polypeptide and protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein,RSV G glycoprotein, influenza virus neuraminidase, influenza virushemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB,gC, gD, and gE) and hepatitis B surface antigen) that is capable ofeliciting an immune response. Antibodies useful in this invention fortreatment of a viral infectious disease include, but are not limited to,antibodies against antigens of pathogenic viruses, including as examplesand not by limitation: adenovirdiae (e.g., mastadenovirus andaviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpessimplex virus 2, herpes simplex virus 5, and herpes simplex virus 6),leviviridae (e.g., levivirus, enterobacteria phase MS2, allolevirus),poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus,capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, andentomopoxyirinae), papovaviridae (e.g., polyomavirus andpapillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenzavirus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumpsvirus), pneumonovirinae (e.g., pneumovirus, human respiratory synctialvirus), and metapneumovirus (e.g., avian pneumovirus and humanmetapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus,hepatovirus (e.g., human hepatits A virus), cardiovirus, andapthovirus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus,cypovirus, fijivirus, phytoreovirus, and oryzavirus), retroviridae(e.g., mammalian type B retroviruses, mammalian type C retroviruses,avian type C retroviruses, type D retrovirus group, BLV-HTLVretroviruses, lentivirus (e.g., human immunodeficiency virus 1 and humanimmunodeficiency virus 2), spumavirus), flaviviridae (e.g., hepatitis Cvirus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g.,alphavirus (e.g., sindbis virus) and rubivirus (e.g., rubella virus)),rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus,cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus,lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), andcoronaviridae (e.g., coronavirus and torovirus).

Specific examples of antibodies available useful for the treatment of aviral infectious disease include, but are not limited to, PRO542(Progenics) which is a CD4 fusion antibody useful for the treatment ofHIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; and Protovir(Protein Design Labs, Inc., CA) which is a humanized IgG1 antibodyuseful for the treatment of cytomegalovirus (CMV); and palivizumab(SYNAGIS®; MedImmune, Inc.; International Publication No. WO 02/43660)which is a humanized antibody useful for treatment of RSV.

In a specific embodiment, the anti-viral agents used in the compositionsand methods of the invention inhibit or reduce a pulmonary orrespiratory virus infection, inhibit or reduce the replication of avirus that causes a pulmonary or respiratory infection, or inhibit orreduce the spread of a virus that causes a pulmonary or respiratoryinfection to other cells or subjects. In another preferred embodiment,the anti-viral agents used in the compositions and methods of theinvention inhibit or reduce infection by RSV, hMPV, or PIV, inhibit orreduce the replication of RSV, hMPV, or PIV, or inhibit or reduce thespread of RSV, hMPV, or PIV to other cells or subjects. Examples of suchagents and methods of treatment of RSV, hMPV, and/or PIV infectionsinclude, but are not limited to, nucleoside analogs, such as zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir,indinavir, ritonavir, and the alpha-interferons. See U.S. Prov. PatentApp. No. 60/398,475 filed Jul. 25, 2002, entitled “Methods of Treatingand Preventing RSV, HMPV, and PIV Using Anti-RSV, Anti-HMPV, andAnti-PIV Antibodies,” and U.S. patent application Ser. No. 10/371,122filed Feb. 21, 2003, which are incorporated herein by reference in itsentirety.

In preferred embodiments, the viral infection is RSV and the anti-viralantigen is an antibody that immunospecifically binds to an antigen ofRSV. In certain embodiments, the anti-RSV-antigen antibody bindsimmunospecifically to an RSV antigen of the Group A of RSV. In otherembodiments, the anti-RSV-antigen antibody binds immunospecifically toan RSV antigen of the Group B of RSV. In other embodiments, an antibodybinds to an antigen of RSV of one Group and cross reacts with theanalogous antigen of the other Group. In particular embodiments, theanti-RSV-antigen antibody binds immunospecifically to a RSVnucleoprotein, RSV phosphoprotein, RSV matrix protein, RSV smallhydrophobic protein, RSV RNA-dependent RNA polymerase, RSV F protein,and/or RSV G protein. In additional specific embodiments, theanti-RSV-antigen antibody binds to allelic variants of a RSVnucleoprotein, a RSV nucleocapsid protein, a RSV phosphoprotein, a RSVmatrix protein, a RSV attachment glycoprotein, a RSV fusionglycoprotein, a RSV nucleocapsid protein, a RSV matrix protein, a RSVsmall hydrophobic protein, a RSV RNA-dependent RNA polymerase, a RSV Fprotein, a RSV L protein, a RSV P protein, and/or a RSV G protein.

It should be recognized that antibodies that immunospecifically bind toa RSV antigen are known in the art. For example, SYNAGIS® (Palivizumab)is a humanized monoclonal antibody presently used for the prevention ofRSV infection in pediatric patients. In a specific embodiment, anantibody to be used with the methods of the present invention ispalivizumab or an antibody-binding fragment thereof (e.g., a fragmentcontaining one or more complementarity determining regions (CDRs) andpreferably, the variable domain of palivizumab). The amino acid sequenceof palivizumab is disclosed, e.g., in Johnson et al., 1997, J.Infectious Disease 176:1215-1224, and U.S. Pat. No. 5,824,307 andInternational Application Publication No.: WO 02/43660, entitled“Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis andTreatment”, by Young et al., which are incorporated herein by referencein their entireties.

One or more antibodies or antigen-binding fragments thereof that bindimmunospecifically to a RSV antigen comprise a Fc domain with a higheraffinity for the FcRn receptor than the Fc domain of palivizumab canalso be used in accordance with the invention. Such antibodies aredescribed in U.S. patent application Ser. No. 10/020,354, filed Dec. 12,2001, which is incorporated herein by reference in its entireties.Further, the anti-RSV-antigen antibody A4B4; P12f2 P12f4; P11d4; Ale9;A12a6; A13c4; A17d4; A4B4; 1X-493L1; FR H3-3F4; M3H9; Y10H6; DG; AFFF;AFFF(1); 6H8; L1-7E5; L2-15B10; A13a11; A1h5; A4B4(1); A4B4-F52S; orA4B4L1FR-S28R can be used in accordance with the invention. Theseantibodies are disclosed in International Application Publication No.:WO 02/43660, entitled “Methods of Administering/Dosing Anti-RSVAntibodies for Prophylaxis and Treatment”, by Young et al., and U.S.Provisional Patent Application 60/398,475 filed Jul. 25, 2002, entitled“Methods of Treating and Preventing RSV, HMPV, and PIV Using Anti-RSV,Anti-HMPV, and Anti-PIV Antibodies” which are incorporated herein byreference in their

In certain embodiments, the anti-RSV-antigen antibodies are theanti-RSV-antigen antibodies of or are prepared by the methods of U.S.application Ser. No. 09/724,531, filed Nov. 28, 2000; Ser. No.09/996,288, filed Nov. 28, 2001; and Ser. No. 09/996,265, filed Nov. 28,2001, all entitled “Methods of Administering/Dosing Anti-RSV Antibodiesfor Prophylaxis and Treatment”, by Young et al., which are incorporatedby reference herein in their entireties. Methods and composition forstabilized antibody formulations that can be used in the methods of thepresent invention are disclosed in U.S. Provisional Application Nos.:60/388,921, filed Jun. 14, 2002, and 60/388,920, filed Jun. 14, 2002,which are incorporated by reference herein in their entireties.

Anti-viral therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (59^(th) ed., 2005).Additional information on respiratory viral infections is available inCecil Textbook of Medicine (18th ed., 1988).

5.5.5.1. Therapies for Viral Infections

In a specific embodiment, the invention provides methods of preventing,treating, managing, and/or ameliorating a viral respiratory infection orone or more symptoms thereof, said method comprising administering to asubject in need thereof an effective amount of one or more antibodyformulations of the invention. In another embodiment, the inventionprovides a method of preventing, treating, managing, and/or amelioratinga viral respiratory infection or one or more symptoms thereof, saidmethod comprising administering to a subject in need thereof aneffective amount of one or more antibody formulations of the inventionand an effective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other the than antibody formulationsof the invention.

In certain embodiments, an effective amount of one or more antibodyformulations of the invention is administered in combination with aneffective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) currently being used, have beenused, or are known to be useful in the prevention, management,treatment, and/or amelioration of a viral infection, preferably a viralrespiratory infection, or one or more symptoms thereof to a subject inneed thereof. Therapies for a viral infection, preferably a viralrespiratory infection include, but are not limited to, anti-viral agentssuch as amantadine, oseltamivir, ribaviran, palivizumab (SYNAGIS™), andanamivir. In certain embodiments, an effective amount of one or moreantibody formulations of the invention is administered in combinationwith one or more supportive measures to a subject in need thereof toprevent, manage, treat, and/or ameliorate a viral infection or one ormore symptoms thereof. Non-limiting examples of supportive measuresinclude humidification of the air by an ultrasonic nebulizer, aerolizedracemic epinephrine, oral dexamethasone, intravenous fluids, intubation,fever reducers (e.g., ibuprofen, acetometaphin), and antibiotic and/oranti-fungal therapy (i.e., to prevent or treat secondary bacterialinfections).

Any type of viral infection or condition resulting from or associatedwith a viral infection (e.g., a respiratory condition) can be prevented,treated, managed, and/or ameliorated in accordance with the methods ofthe invention, said methods comprising administering an effective amountof one or more antibody formulations of the invention alone or incombination with an effective amount of another therapy (e.g., aprophylactic or therapeutic agent other than antibody formulations ofthe invention). Examples of viruses which cause viral infectionsinclude, but are not limited to, retroviruses (e.g., human T-celllymphotrophic virus (HTLV) types I and II and human immunodeficiencyvirus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types Iand II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenavirues(e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus virus,human respiratory syncytial virus, mumps, hMPV, and pneumovirus),adenoviruses, bunyaviruses (e.g., hantavirus), comaviruses, filoviruses(e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellowfever virus, and Japanese encephalitis virus), hepadnaviruses (e.g.,hepatitis B viruses (HBV)), orthomyoviruses (e.g., influenza viruses A,B and C and PIV), papovaviruses (e.g., papillomavirues), picomaviruses(e.g., rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses,reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus), andrhabdoviruses (e.g., rabies virus). Biological responses to a viralinfection include, but not limited to, elevated levels of IgEantibodies, increased proliferation and/or infiltration of T cells,increased proliferation and/or infiltration of B cells, epithelialhyperplasia, and mucin production. In a specific embodiment, theinvention also provides methods of preventing, treating, managing,and/or ameliorating viral respiratory infections that are associatedwith or cause the common cold, viral pharyngitis, viral laryngitis,viral croup, viral bronchitis, influenza, parainfluenza viral diseases(“PIV”) diseases (e.g., croup, bronchiolitis, bronchitis, pneumonia),respiratory syncytial virus (“RSV”) diseases, metapneumavirus diseases,and adenovirus diseases (e.g., febrile respiratory disease, croup,bronchitis, pneumonia), said method comprising administering aneffective amount of one or more antibody formulations of the inventionalone or in combination with an effective amount of another therapy.

In a specific embodiment, influenza virus infections, PIV infections,hMPV infections, adenovirus infections, and/or RSV infections, or one ormore of symptoms thereof are prevented, treated, managed, and/and/orameliorated in accordance with the methods of the invention. In aspecific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating a RSV respiratory infection orone or more symptoms thereof, said methods comprising administering to asubject in need thereof an effective amount of one or more antibodyformulations of the invention alone or in combination with one or moreanti-viral agents such as, but not limited to, amantadine, rimantadine,oseltamivir, znamivir, ribaviran, RSV-IVIG (i.e., intravenous immuneglobulin infusion) (RESPIGAM™), and palivizumab (SYNAGIS™). In aspecific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating a PIV infection or one or moresymptoms thereof, said methods comprising administering to a subject inneed thereof an effective amount of one or more antibody formulations ofthe invention alone or in combination with an effective amount of one ormore anti-viral agents such as, but not limited to, amantadine,rimantadine, oseltamivir, znamivir, ribaviran, and palivizumab(SYNAGIS™). In another specific embodiment, the invention providesmethods for preventing, treating, managing, and/or ameliorating a hMPVinfection or one or more symptoms thereof, said methods comprising ofadministering an effective amount of one or more antibody formulationsof the invention alone or in combination with an effective amount of oneor more anti-viral agents, such as, but not limited to, amantadine,rimantadine, oseltamivir, znamivir, ribaviran, and palivizumab(SYNAGIS™) to a subject in need thereof. In a specific embodiment, theinvention provides methods for preventing, treating, managing, and/orameliorating influenza, said methods comprising administering aneffective amount of one or more antibody formulations of the inventionalone or in combination with an effective amount of an anti-viral agentsuch as, but not limited to zanamivir (RELENZA®), oseltamivir(TAMIFLU®), rimantadine, and amantadine (SYMADINE®; SYMMETREL®) to asubject in need thereof.

The invention provides methods for preventing the development of asthmain a subject who suffers from or had suffered from a viral respiratoryinfection, said methods comprising adminsitering an effective amount ofone or more antibody formulations of the invention alone or incombination with an effective amount of another therapy. In a specificembodiment, the subject is an infant born prematurely, an infant, or achild. In another specific embodiment, the subject suffered from orsuffers from RSV infection.

In a specific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating one or more secondary responsesto a primary viral infection, said methods comprising of administeringan effective amount of one or more antibody formulations of theinvention alone or in combination with an effective amount of othertherapies (e.g., other prophylactic or therapeutic agents). Examples ofsecondary responses to a primary viral infection, particularly a primaryviral respiratory infection, include, but are not limited to,asthma-like responsiveness to mucosal stimula, elevated totalrespiratory resistance, increased susceptibility to secondary viral,bacterial, and fungal infections, and development of such conditionssuch as, but not limited to, pneumonia, croup, and febrile bronchitis.

In a specific embodiment, the invention provides methods of preventing,treating, managing, and/or ameliorating a viral infection or one or moresymptoms thereof, said methods comprising administering to a subject inneed thereof an effective amount of one or more antibody formulations ofthe invention in combination with an effective amount of VITAXIN™(MedImmune, Inc., International Publication No. WO 00/78815,International Publication No. WO 02/070007 A1, dated Sep. 12, 2002,entitled “Methods of Preventing or Treating Inflammatory or AutoimmuneDisorders by Administering Integrin AlphaV Beta3 Antagonists,”International Publication No. WO 03/075957 A1, dated Sep. 18, 2003,entitled “The Prevention or Treatment of Cancer Using IntegrinAlphaVBeta3 Antagonists in Combination With Other Agents,” U.S. PatentPub. No. U.S. 2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods ofPreventing or Treating Inflammatory or Autoimmune Disorders byAdministering Integrin α_(v)β3 Antagonists in Combination With OtherProphylactic or Therapeutic Agents,” and International Publication No.WO 03/075741 A2, dated Sep. 18, 2003, entitled, “Methods of Preventingor Treating Disorders by Administering an Integrin αvβ3 Antagonist inCombination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate,”each of which is incorporated herewith by reference in its entirety). Inanother specific embodiment, the invention provides methods forpreventing, treating, managing, and/or ameliorating a viral infection orone or more symptoms thereof, said methods comprising administering to asubject in need thereof an effective amount of one or more antibodies ofthe invention in combination with an effective amount of siplizumab(MedImmune, Inc., International Pub. No. WO 02/069904). In anotherembodiment, the invention provides methods for preventing, treating,managing, and/or ameliorating a viral infection or one or more symptomsthereof, said methods comprising administering to a subject in needthereof an effective amount of one or more antibodies of the inventionin combination with an effective amount of one or more EphA2 inhibitors(e.g., one or more anti-EphA2 antibodies (MedImmune, Inc.; InternationalPublication No. WO 02/102974 A4, dated Dec. 27, 2002, entitled “MutantProteins, High Potency Inhibitory Antibodies and FIMCH CrystalStructure,” International Publication No. 03/094859 A2, dated Nov. 20,2003, entitled “EphA2 Monoclonal Antibodies and Methods of Use Thereof,”U.S. application Ser. No. 10/436,783; and U.S. application Ser. No.60/379,368, each of which is incorporated herewith by reference)). Inyet another embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating a viral infection or one or moresymptoms thereof, said methods comprising administering to a subject inneed thereof an effective amount of one or more antibodies of theinvention in combination with an effective amount of VITAXIN™,siplizumab, and/or EphA2.

In one embodiment, an effective amount of one or more antibodyformulations of the invention is administered in combination with aneffective amount of one or more anti-IgE antibodies to a subject toprevent, treat, manage, and/or ameliorate a viral infection or one ormore symptoms thereof. In a specific embodiment, an effective amount ofone or more antibody formulations of the invention is administered incombination with an effective amount of anti-IgE antibody TNX901 to asubject to prevent, treat, manage, and/or ameliorate a viral infectionor one or more symptoms thereof. In a specific embodiment, an effectiveamount of one or more antibody formulations of the invention isadministered in combination with an effective amount of anti-IgEantibody rhuMAb-E25 omalizumab to a subject to prevent, treat, manage,and/or ameliorate a viral infection or one or more symptoms thereof. Inanother embodiment, an effective amount of one or more antibodyformulations of the invention is administered in combination with aneffective amount of anti-IgE antibody HMK-12 to a subject to prevent,treat, manage, and/or ameliorate a viral infection or one or moresymptoms thereof. In a specific embodiment, an effective amount of oneor more antibody formulations of the invention is administered incombination with an effective amount of anti-IgE antibody 6HD5 to asubject to prevent, treat, manage, and/or ameliorate a viral infectionor one or more symptoms thereof. In another embodiment, an effectiveamount of one or more antibody formulations of the invention isadministered in combination with an effective amount of anti-IgEantibody MAb Hu-901 to a subject to prevent, treat, manage, and/orameliorate a viral infection or one or more symptoms thereof.

The invention encompasses methods for preventing the development ofviral infections, preferably viral respiratory infections, in a patientexpected to suffer from a viral infection or at increased risk of suchan infection, e.g., patients with suppressed immune systems (e.g.,organ-transplant recipients, AIDS patients, patients undergoingchemotherapy, the elderly, infants born prematurely, infants, children,patients with carcinoma of the esophagus with obstruction, patients withtracheobronchial fistula, patients with neurological diseases (e.g.,caused by stroke, amyotrophic lateral sclerosis, multiple sclerosis, andmyopathies), and patients already suffering from a respiratoryinfection). The patients may or may not have been previously treated fora respiratory infection.

The antibody formulations of the invention or combination therapies ofthe invention may be used as the first, second, third, fourth, or fifththerapy to prevent, manage, treat, and/or ameliorate a viral infection,preferably a viral respiratory infection, or one or more symptomthereof. The invention also includes methods of preventing, treating,managing, and/or ameliorating a viral infection, preferably a viralrespiratory infection, or one or more symptoms thereof in a patientundergoing therapies for other diseases or disorders associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, diseases or disorders associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, autoimmune diseases, inflammatory diseases, proliferativediseases, or infections (preferably, respiratory infections), or one ormore symptoms thereof. The invention encompasses methods of preventing,managing, treating, and/or ameliorating a viral infection, preferably aviral respiratory infection, or one or more symptoms thereof in apatient before any adverse effects or intolerance to therapies otherthan antibody formulations of the invention develops. The invention alsoencompasses methods of preventing, treating, managing, and/orameliorating a viral infection, preferably a viral respiratoryinfection, or a symptom thereof in refractory patients. In certainembodiments, a patient with a viral infection, preferably a viralrespiratory infection, is refractory to a therapy when the infection hasnot significantly been eradicated and/or the symptoms have not beensignificantly alleviated. The determination of whether a patient isrefractory can be made either in vivo or in vitro by any method known inthe art for assaying the effectiveness of a treatment of infections,using art-accepted meanings of “refractory” in such a context. Invarious embodiments, a patient with a viral respiratory infection isrefractory when viral replication has not decreased or has increased.The invention also encompasses methods of preventing the onset orreoccurrence of viral respiratory infections in patients at risk ofdeveloping such infections. The invention also encompasses methods ofpreventing, managing, treating, and/or ameliorating a viral infection,preferably a viral respiratory infection, or a symptom thereof inpatients who are susceptible to adverse reactions to conventionaltherapies. The invention further encompasses methods for preventing,treating, managing, and/or ameliorating a viral infection, preferably aviral respiratory infection, for which no anti-viral therapy isavailable.

The invention encompasses methods for preventing, treating, managing,and/or ameliorating a viral infection, preferably a viral respiratoryinfection, or a symptom thereof in a patient who has proven refractoryto therapies other than antibody formulations of the invention but areno longer on these therapies. In certain embodiments, the patients beingmanaged or treated in accordance with the methods of this invention arepatients already being treated with antibiotics, anti-virals,anti-fungals, or other biological therapy/immunotherapy. Among thesepatients are refractory patients, patients who are too young forconventional therapies, and patients with reoccurring viral infectionsdespite management or treatment with existing therapies.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating a viral infection, preferably a viralrespiratory infection, or one or more symptoms thereof as an alternativeto other conventional therapies. In specific embodiments, the patientbeing managed or treated in accordance with the methods of the inventionis refractory to other therapies or is susceptible to adverse reactionsfrom such therapies. The patient may be a person with a suppressedimmune system (e.g., post-operative patients, chemotherapy patients, andpatients with immunodeficiency disease), a person with impaired renal orliver function, the elderly, children, infants, infants bornprematurely, persons with neuropsychiatric disorders or those who takepsychotropic drugs, persons with histories of seizures, or persons onmedication that would negatively interact with conventional agents usedto prevent, manage, treat, and/or ameliorate a viral infection or one ormore symptoms thereof.

Viral infection therapies and their dosages, routes of administrationand recommended usage are known in the art and have been described insuch literature as the Physician's Desk Reference (59th ed., 2005).

5.5.6. Bacterial Infections

The invention provides a method of preventing, treating, managing,and/or ameliorating a bacterial infection or one or more symptomsthereof, said method comprising administering to a subject in needthereof an effective amount of one or more antibody formulations of theinvention. In another embodiment, the invention provides a method ofpreventing, treating, managing, and/or ameliorating a bacterialinfection or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof an effective amount of a oneor more antibody formulations of the invention and an effective amountof one or more therapies (e.g., one or more prophylactic or therapeuticagents), other than antibody formulations of the invention.Anti-bacterial agents and therapies well known to one of skill in theart for the prevention, treatment, management, or amelioration ofbacterial infections can be used in the compositions and methods of theinvention. Non-limiting examples of anti-bacterial agents includeproteins, polypeptides, peptides, fusion proteins, antibodies, nucleicacid molecules, organic molecules, inorganic molecules, and smallmolecules that inhibit or reduce a bacterial infection, inhibit orreduce the replication of bacteria, or inhibit or reduce the spread ofbacteria to other subjects. In particular, examples of anti-bacterialagents include, but are not limited to, penicillin, cephalosporin,imipenem, axtreonam, vancomycin, cycloserine, bacitracin,chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin,tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin,trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B,nystatin, ketocanazole, isoniazid, metronidazole, and pentamidine.

In a preferred embodiment, the anti-bacterial agent is an agent thatinhibits or reduces a pulmonary or respiratory bacterial infection,inhibits or reduces the replication of a bacteria that causes apulmonary or respiratory infection, or inhibits or reduces the spread ofa bacteria that causes a pulmonary or respiratory infection to othersubjects. In cases in which the pulmonary or respiratory bacterialinfection is a mycoplasma infection (e.g., pharyngitis,tracheobronchitis, and pneumonia), the anti-bacterial agent ispreferably a tetracycline, erythromycin, or spectinomycin. In cases inwhich the pulmonary or respiratory bacterial infection is pneumoniacaused by an aerobic gram negative bacilli (GNB), the anti-bacterialagent is preferably penicillin, first second, or third generationcephalosporin (e.g., cefaclor, cefadroxil, cephalexin, or cephazolin),erythomycin, clindamycin, an aminoglycoside (e.g., gentamicin,tobramycin, or amikacine), or a monolactam (e.g., aztreonam). In casesin which the pulmonary or respiratory bacterial infection istuberculosis, the anti-bacterial agent is preferably, rifampcin,isonaizid, pyranzinamide, ethambutol, and streptomycin. In cases inwhich the respiratory infection is recurrent aspiration pneumonia, theanti-bacterial agent is preferably penicillin, an aminoglycoside, or asecond or third generation cephalosporin.

5.5.6.1. Therapies for Bacterial Infections

Any type of bacterial infection or condition resulting from orassociated with a bacterial infection (e.g., a respiratory infection)can be prevented, treated, managed, and/or ameliorated in accordancewith the methods of invention. Examples of bacteria which causebacterial infections include, but not limited to, the Aquaspirillumfamily, Azospirillum family, Azotobacteraceae family, Bacteroidaceaefamily, Bartonella species, Bdellovibrio family, Campylobacter species,Chlamydia species (e.g., Chlamydia pneumoniae), clostridium,Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella,Enterobacter aerogenes, Erwinia species, Escherichia coli, Hafniaspecies, Klebsiella species, Morganella species, Proteus vulgaris,Providencia, Salmonella species, Serratia marcescens, and Shigellaflexneri), Gardinella family, Haemophilus influenzae, Halobacteriaceaefamily, Helicobacter family, Legionallaceae family, Listeria species,Methylococcaceae family, mycobacteria (e.g., Mycobacteriumtuberculosis), Neisseriaceae family, Oceanospirillum family,Pasteurellaceae family, Pneumococcus species, Pseudomonas species,Rhizobiaceae family, Spirillum family, Spirosomaceae family,Staphylococcus (e.g., methicillin resistant Staphylococcus aureus andStaphylococcus pyrogenes), Streptococcus (e.g., Streptococcusenteritidis, Streptococcus fasciae, and Streptococcus pneumoniae),Vampirovibr Helicobacter family, and Vampirovibrio family.

In a specific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating a bacterial respiratoryinfection or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof an effective amount of one ormore antibody formulations of the invention. In another embodiment, theinvention provides a method of preventing, treating, managing, and/orameliorating a bacterial respiratory infection or one or more symptomsthereof, said method comprising administering to a subject in needthereof an effective amount of a one or more antibody formulations ofthe invention and an effective amount of one or more therapies (e.g.,prophylactic or therapeutic agents), other than antibody formulations ofthe invention.

In certain embodiments, the invention provides methods to prevent,treat, manage, and/or ameliorate a bacterial infection, preferably abacterial respiratory infection, or one or more of the symptoms, saidmethods comprising administering to a subject in need thereof one ormore antibody formulations of the invention in combination with andeffective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents), other than antibody formulations ofthe invention, used to prevent, treat, manage, and/or amelioratebacterial infections. Therapies for bacterial infections, particularly,bacterial respiratory infections include, but are not limited to,anti-bacterial agents (e.g., aminoglycosides (e.g., gentamicin,tobramycin, amikacin, netilimicin) aztreonam, celphalosporins (e.g.,cefaclor, cefadroxil, cephalexin, cephazolin), clindamycin,erythromycin, penicillin (e.g., penicillin V, crystalline penicillin G,procaine penicillin G), spectinomycin, and tetracycline (e.g.,chlortetracycline, doxycycline, oxytetracycine)) and supportiverespiratory therapy, such as supplemental and mechanical ventilation. Incertain embodiments, one or more antibody formulations of the inventionare administered in combination with one or more supportive measures toa subject in need thereof to prevent, manage, treat, and/or ameliorate abacterial infection or one or more symptoms thereof. Non-limitingexamples of supportive measures include humidification of air byultrasonic nebulizer, aerolized racemic epinephrine, oral dexamethasone,intravenous fluids, intubation, fever reducers (e.g., ibuprofen,acetometaphin), and more preferably, antibiotic or anti-viral therapy(i.e., to prevent or treat secondary infections).

The invention provides methods for preventing, managing, treating,and/or ameliorating a biological response to a bacterial infection,preferably a bacterial respiratory infection, such as, but not limitedto, elevated levels of IgE antibodies, mast cell proliferation,degranulation, and/or infiltration, increased proliferation and/orinfiltration of B cells, and increased proliferation and/or infiltrationof T cells, said methods comprising administering to a subject in needthereof an effective amount of one or more antibody formulations of theinvention alone or in combination with an effective amount one or moretherapies (e.g. a prophylactic or therapeutic agent) other than antibodyformulations of the invention. The invention also provides methods ofpreventing, treating, managing, and/or ameliorating respiratoryconditions caused by or associated with bacterial infections, preferablybacterial respiratory infections, such as, but not limited to,pneumonococcal pneumonia, pneumonia caused by aerobic gram-negativebacilli, recurrent aspiration pneumonia, legionellosis, streptococcaldisease, infections caused by Hemophilus, whooping cough, meningitis, ortuberculosis, said methods comprising administering to a subject in needthereof an effective amount of one or more antibody formulations of theinvention alone or in combination with an effective amount of anothertherapy.

In a specific embodiment, the methods of the invention are utilized toprevent, treat, manage, and/or ameliorate a bacterial respiratoryinfection caused by Pneumonococcus, Mycobacteria, aerobic gram-negativebacilli, Streptococcus, or Hemophilus or one or more symptoms thereof,said method comprising administering to a subject in need thereof of aneffective amount of one or more antibody formulations of the inventionalone or in combination with an effective amount of one or more othertherapies (e.g., one or more prophylactic or therapeutic agents) otherthan antibody formulations of the invention.

In a specific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating one or more secondary conditionsor responses to a primary bacterial infection, preferably a primarybacterial respiratory infection, said method comprising administering toa subject in need thereof an effective amount of one or more antibodyformulations of the invention alone or in combination with an effectiveamount of other therapies (e.g., other prophylactic or therapeuticagents). Examples of secondary conditions or responses to a primarybacterial infection, particularly a bacterial respiratory infection,include, but are not limited to, asthma-like responsiveness to mucosalstimula, elevated total respiratory resistance, increased susceptibilityto secondary viral, bacterial, and fungal infections, and development ofsuch conditions such as, but not limited to, pneumonia, croup, andfebrile bronchitits.

In a specific embodiment, the methods of the invention are used toprevent, manage, treat, and/or ameliorate a bacterial infection,preferably a bacterial respiratory infection, or one or more symptomsthereof, said methods comprising administering to a subject in needthereof an effective amount of one or more antibodies of the inventionin combination with an effective amount of VITAXIN™ (MedImmune, Inc.,International Publication No. WO 00/78815, International Publication No.WO 02/070007 A1, dated Sep. 12, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering IntegrinAlphaV Beta3 Antagonists,” International Publication No. WO 03/075957A1, dated Sep. 18, 2003, entitled “The Prevention or Treatment of CancerUsing Integrin AlphaVBeta3 Antagonists in Combination With OtherAgents,” U.S. Patent Pub. No. U.S. 2002/0168360 A1, dated Nov. 14, 2002,entitled “Methods of Preventing or Treating Inflammatory or AutoimmuneDisorders by Administering Integrin α_(v)β3 Antagonists in CombinationWith Other Prophylactic or Therapeutic Agents,” and InternationalPublication No. WO 03/075741 A2, dated Sep. 18, 2003, entitled, “Methodsof Preventing or Treating Disorders by Administering an Integrin αvβ3Antagonist in Combination With an HMG-CoA Reductase Inhibitor or aBisphosphonate,” each of which is incorporated herewith by reference inits entirety). In another specific embodiment, the methods of theinvention are used to prevent, manage, treat, and/or ameliorate abacterial infection, preferably a bacterial respiratory infection, orone or more symptoms thereof, said methods comprising administering to asubject in need thereof an effective amount of one or more antibodies ofthe invention in combination with an effective amount of siplizumab(MedImmune, Inc., International Pub. No. WO 02/069904). In anotherembodiment, the methods of the invention are used to prevent, manage,treat, and/or ameliorate a bacterial infection, preferably a bacterialrespiratory infection, or one or more symptoms thereof, said methodscomprising administering to a subject in need thereof an effectiveamount of one or more antibodies of the invention in combination with aneffective amount of one or more EphA2 inhibitors (e.g., one or moreanti-EphA2 antibodies (MedImmune, Inc.; International Publication No. WO02/102974 A4, dated Dec. 27, 2002, entitled “Mutant Proteins, HighPotency Inhibitory Antibodies and FIMCH Crystal Structure,”International Publication No. 03/094859 A2, dated Nov. 20, 2003,entitled “EphA2 Monoclonal Antibodies and Methods of Use Thereof,” U.S.Application Ser. No. 10/436,783; and U.S. Appn. No. 60/379,368, each ofwhich is incorporated herewith by reference)). In yet anotherembodiment, the invention provides methods of preventing, treating,managing, and/or ameliorating a bacterial infection, preferably abacterial respiratory infection, or one or more symptoms thereof, saidmethods comprising administering an effective amount of one or moreantibodies of the invention in combination with an effective amount ofVITAXIN™, siplizumab, and/or EphA2.

The invention encompasses methods for preventing the development ofbacterial infections, preferably bacterial respiratory infections, in apatient expected to suffer from a bacterial respiratory infection or atincreased risk of such an infection, e.g., patients with suppressedimmune systems (e.g., organ-transplant recipients, AIDS patients,patients undergoing chemotherapy, the elderly, infants born prematurely,infants, children, patients with carcinoma of the esophagus withobstruction, patients with tracheobronchial fistula, patients withneurological diseases (e.g., caused by stroke, amyotrophic lateralsclerosis, multiple sclerosis, and myopathies), and patients alreadysuffering from an infection, particularly a respiratory infection). Thepatients may or may not have been previously treated for an infection.

The antibody formulations of the invention or combination therapies ofthe invention may be used as the first, second, third, fourth, or fifththerapy to prevent, manage, treat, and/or ameliorate a bacterialinfection, preferably a bacterial respiratory infection, or one or moresymptom thereof. The invention also includes methods of preventing,treating, managing, and/or ameliorating a bacterial infection,preferably a bacterial respiratory infection, or one or more symptomsthereof in a patient undergoing therapies for other diseases ordisorders. The invention encompasses methods of preventing, managing,treating, and/or ameliorating a bacterial infection, preferably abacterial respiratory infection, or one or more symptoms thereof in apatient before any adverse effects or intolerance to therapies otherthan antibody formulations of the invention develops. The invention alsoencompasses methods of preventing, treating, managing, and/orameliorating a bacterial infection, preferably a bacterial respiratoryinfection, or a symptom thereof in refractory patients. In certainembodiments, a patient with a bacterial respiratory infection isrefractory to a therapy when the infection has not significantly beeneradicated and/or the symptoms have not been significantly alleviated.The determination of whether a patient is refractory can be made eitherin vivo or in vitro by any method known in the art for assaying theeffectiveness of a treatment of infections, using art-accepted meaningsof “refractory” in such a context. In various embodiments, a patientwith a bacterial respiratory infection is refractory when bacterialreplication has not decreased or has increased. The invention alsoencompasses methods of preventing the onset or reoccurrence of abacterial infection, preferably a bacterial respiratory infection, inpatients at risk of developing such infection. The invention alsoencompasses methods of preventing, managing, treating, and/orameliorating a bacterial infection, preferably a bacterial respiratoryinfection, or a symptom thereof in patients who are susceptible toadverse reactions to conventional therapies. The invention furtherencompasses methods for preventing, treating, managing, and/orameliorating bacterial infections, preferably bacterial respiratoryinfections, for which no anti-bacterial therapy is available.

The invention encompasses methods for preventing, treating, managing,and/or ameliorating a bacterial infection, preferably a bacterialrespiratory infection, or a symptom thereof in a patient who has provenrefractory to therapies other than antibody formulations of theinvention, but are no longer on these therapies. In certain embodiments,the patients being managed or treated in accordance with the methods ofthis invention are patients already being treated with anti-inflammatoryagents, antibiotics, anti-virals, anti-fungals, or other biologicaltherapy/immunotherapy. Among these patients are refractory patients,patients who are too young for conventional therapies, and patients withreoccurring bacterial infections despite management or treatment withexisting therapies.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating a bacterial infection, preferably abacterial respiratory infection, or one or more symptoms thereof as analternative to other conventional therapies. In specific embodiments,the patient being managed or treated in accordance with the methods ofthe invention is refractory to other therapies or is susceptible toadverse reactions from such therapies. The patient may be a person witha suppressed immune system (e.g., post-operative patients, chemotherapypatients, and patients with immunodeficiency disease), a person withimpaired renal or liver function, the elderly, children, infants,infants born prematurely, persons with neuropsychiatric disorders orthose who take psychotropic drugs, persons with histories of seizures,or persons on medication that would negatively interact withconventional agents used to prevent, manage, treat, and/or ameliorate abacterial infection, preferably a bacterial respiratory infection, orone or more symptoms thereof.

Bacterial infection therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (59thed., 2005).

5.5.7. Fungal Infections

Anti-fungal agents and therapies well known to one of skill in the artfor prevention, management, treatment, and/or amelioration of a fungalinfection or one or more symptoms thereof (e.g., a fungal respiratoryinfection) can be used in the compositions and methods of the invention.Non-limiting examples of anti-fungal agents include proteins,polypeptides, peptides, fusion proteins, antibodies, nucleic acidmolecules, organic molecules, inorganic molecules, and small moleculesthat inhibit and/or reduce fungal infection, inhibit and/or reduce thereplication of fungi, or inhibit and/or reduce the spread of fungi toother subjects. Specific examples of anti-fungal agents include, but arenot limited to, azole drugs (e.g., miconazole, ketoconazole (NIZORAL®),caspofungin acetate (CANCIDAS®), imidazole, triazoles (e.g., fluconazole(DIFLUCAN®)), and itraconazole (SPORANOX®)), polyene (e.g., nystatin,amphotericin B (FUNGIZONE®), amphotericin B lipid complex(“ABLC”)(ABELCET®), amphotericin B colloidal dispersion(“ABCD”)(AMPHOTEC®), liposomal amphotericin B (AMBISONE®)), potassiumiodide (KI), pyrimidine (e.g., flucytosine (ANCOBON®)), and voriconazole(VFEND®). See, e.g., Table 4 for a list of specific anti-fungal agentsand their recommended dosages.

TABLE 4 Anti-fungal Agents Anti-fungal Agent Dosage Amphotericin BABELCET ® (lipid complex injection) 5 mg/kg/day AMBISOME ® (liposome forinjection) 3-5 mg/kg/day AMPHOTEC ® (complex for injection) 3-4mg/kg/day Caspofungin acetate (CANCIDAS ®) 70 mg on day one followed by50 mg/day Fluconazole (DIFLUCAN ®) up to 400 mg/day (adults) up to 12mg/kg/day (children) Itraconazole (SPORANOX ®) 200-400 mg/dayFlucytosine (ANCOBON ®) 50-150 mg/kg/day in divided dose every 6 hoursLiposomal nystatin 1-4 mg/kg Ketoconazole (NIZORAL ®) 200 mg singledaily dose up to 400 mg/day in two divided doses (adults) 3.3-6.6mg/kg/day for children 2 years old and older Voriconazole (VFEND ®) 6mg/kg i.v. loading dose every 12 hours for two doses, followed bymaintenance dose of 4 mg/kg i.v. every 12 hours, then oral maintenancedose of 200-100 mg tablet

In certain embodiments, the anti-fungal agent is an agent that inhibitsor reduces a respiratory fungal infection, inhibits or reduces thereplication of a fungus that causes a pulmonary or respiratoryinfection, or inhibits or reduces the spread of a fungus that causes apulmonary or respiratory infection to other subjects. In cases in whichthe pulmonary or respiratory fungal infection is Blastomycesdermatitidis, the anti-fungal agent is preferably itraconazole,amphotericin B, fluconazole, or ketoconazole. In cases in which thepulmonary or respiratory fungal infection is pulmonary aspergilloma, theanti-fungal agent is preferably amphotericin B, liposomal amphotericinB, itraconazole, or fluconazole. In cases in which the pulmonary orrespiratory fungal infection is histoplasmosis, the anti-fungal agent ispreferably amphotericin B, itraconazole, fluconazole, or ketoconazole.In cases in which the pulmonary or respiratory fungal infection iscoccidioidomycosis, the anti-fungal agent is preferably fluconazole oramphotericin B. In cases in which the pulmonary or respiratory fungalinfection is cryptococcosis, the anti-fungal agent is preferablyamphotericin B, fluconazole, or combination of the two agents. In casesin which the pulmonary or respiratory fungal infection is chromomycosis,the anti-fungal agent is preferably itraconazole, fluconazole, orflucytosine. In cases in which the pulmonary or respiratory fungalinfection is mucormycosis, the anti-fungal agent is preferablyamphotericin B or liposomal amphotericin B. In cases in which thepulmonary or respiratory fungal infection is pseudoallescheriasis, theanti-fungal agent is preferably itraconazole ore miconazole.

Anti-fungal therapies and their dosages, routes of administration, andrecommended usage are known in the art and have been described in suchliterature as Dodds et al., 2000 Pharmacotherapy 20(11) 1335-1355, thePhysician's Desk Reference (59th ed., 2005) and the Merk Manual ofDiagnosis and Therapy (17th ed., 1999).

5.5.7.1. Anti-fungal Therapies

One or more antibody formulations of the invention can be administeredaccording to methods of the invention to a subject to prevent, treat,manage, and/or ameliorate a fungal infection or one or more symptomsthereof. One or more antibody formulations of the invention may be alsoadministered to a subject to treat, manage, and/or ameliorate a fungalinfection and/or one or more symptoms thereof in combination with one ormore other therapies (e.g., one or more prophylactic or therapeuticagents) other than antibody formulations of the invention which areuseful for the prevention, treatment, management, or amelioration of afungal infection or one or more symptoms thereof.

In a specific embodiment, the invention provides a method of preventing,treating, managing, and/or ameliorating a fungal infection or one ormore symptoms thereof, said method comprising administering to a subjectin need thereof an effective amount of one or more antibody formulationsof the invention. In another embodiment, the invention provides a methodof preventing, treating, managing, and/or ameliorating a fungalinfection or one or more symptoms thereof, said method comprisingadministering to a subject in need thereof an effective amount of a oneor more antibody formulations of the invention and an effective amountof one or more therapies (e.g., prophylactic or therapeutic agents),other than antibody formulations of the invention.

Any type of fungal infection or condition resulting from or associatedwith a fungal infection (e.g., a respiratory infection) can beprevented, treated, managed, and/or ameliorated in accordance with themethods of invention. Examples of fungus which cause fungal infectionsinclude, but not limited to, Absidia species (e.g., Absidia corymbiferaand Absidia ramosa), Aspergillus species, (e.g., Aspergillus flavus,Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, andAspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis,Candida species (e.g., Candida albicans, Candida glabrata, Candida kerr,Candida krusei, Candida parapsilosis, Candida pseudotropicalis, Candidaquillermondii, Candida rugosa, Candida stellatoidea, and Candidatropicalis), Coccidioides immitis, Conidiobolus species, Cryptococcusneoforms, Cunninghamella species, dermatophytes, Histoplasma capsulatum,Microsporum gypseum, Mucor pusillus, Paracoccidioides brasiliensis,Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis carinii,Rhizopus species (e.g., Rhizopus arrhizus, Rhizopus oryzae, and Rhizopusmicrosporus), Saccharomyces species, Sporothrix schenckii, zygomycetes,and classes such as Zygomycetes, Ascomycetes, the Basidiomycetes,Deuteromycetes, and Oomycetes.

In a specific embodiment, the invention provides a method of preventing,treating, managing, and/or ameliorating a fungal respiratory infectionor one or more symptoms thereof, said method comprising administering toa subject in need thereof an effective amount of one or more antibodyformulations of the invention. In another embodiment, the inventionprovides a method of preventing, treating, managing, and/or amelioratinga fungal respiratory infection or one or more symptoms thereof, saidmethod comprising administering to a subject in need thereof aneffective amount of one or more antibody formulations of the inventionand an effective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other than antibody formulations ofthe invention.

In certain embodiments, an effective amount of one or more antibodyformulations is administered in combination with an effective amount ofone or more therapies (e.g., one or more prophylactic or therapeuticagents), other than antibody formulations of the invention, which arecurrently being used, have been used, or are known to be useful in theprevention, management, treatment, or amelioration of a fungalinfection, preferably a fungal respiratory infection, to a subject inneed thereof. Therapies for fungal infections include, but are notlimited to, anti-fungal agents such as azole drugs e.g., miconazole,ketoconazole (NIZORAL®), caspofungin acetate (CANCIDAS®), imidazole,triazoles (e.g., fluconazole (DIFLUCAN®)), and itraconazole (SPORANOX®),polyene (e.g., nystatin, amphotericin B colloidal dispersion(“ABCD”)(AMPHOTEC®), liposomal amphotericin B (AMBISONE®)), postassiumiodide (KI), pyrimidine (e.g., flucytosine (ANCOBON®)), and voriconazole(VFEND®). In certain embodiments, an effective amount of one or moreantibody formulations of the invention are administered in combinationwith one or more supportive measures to a subject in need thereof toprevent, manage, treat, and/or ameliorate a fungal infection or one ormore symptoms thereof. Non-limiting examples of supportive measuresinclude humidification of the air by an ultrasonic nebulizer, aerolizedracemic epinephrine, oral desamethasone, intravenous fluids, intubation,fever reducers (e.g., ibuprofen and acetometaphin), and anti-viral oranti-bacterial therapy (i.e., to prevent or treat secondary viral orbacterial infections).

The invention also provides methods for preventing, managing, treatingand/or ameliorating a biological response to a fungal respiratoryinfection such as, but not limited to, elevated levels of IgEantibodies, elevated nerve growth factor (NGF) levels, mast cellproliferation, degranulation, and/or infiltration, increasedproliferation and/or infiltration of B cells, and increasedproliferation and/or infiltration of T cells, said methods comprisingadministration of an effective amount of one or more antibodyformulations that immunospecifically bind to an IL-9 polypeptide aloneor in combination with one or more other therapies.

In a specific embodiment, the invention provides methods for preventing,treating, managing, and/or ameliorating one or more secondary conditionsor responses to a primary fungal infection, preferably a primary fungalrespiratory infection, said method comprising of administering to asubject in need thereof an effective amount of one or more antibodyformulations of the invention alone or in combination with an effectiveamount of other therapies (e.g., other prophylactic or therapeuticagents) other than antibody formulations of the invention. Examples ofsecondary conditions or responses to a primary fungal infections,particularly primary fungal respiratory infection include, but are notlimited to, asthma-like responsiveness to mucosal stimula, elevatedtotal respiratory resistance, increased susceptibility to secondaryviral, fungal, and fungal infections, and development of such conditionssuch as, but not limited to, pneumonia, croup, and febrile bronchitits.

In a specific embodiment, the invention provides methods to prevent,treat, manage, and/or ameliorate a fungal infection, preferably a fungalrespiratory infection, or one or more symptoms thereof, said methodscomprising administering to a subject in need thereof an effectiveamount of one or more antibodies of the invention in combination with aneffective amount of VITAXIN™ (MedImmune, Inc., International PublicationNo. WO 00/78815, International Publication No. WO 02/070007 A1, datedSep. 12, 2002, entitled “Methods of Preventing or Treating Inflammatoryor Autoimmune Disorders by Administering Integrin AlphaV Beta3Antagonists,” International Publication No. WO 03/075957 A1, dated Sep.18, 2003, entitled “The Prevention or Treatment of Cancer Using IntegrinAlphaVBeta3 Antagonists in Combination With Other Agents,” U.S. PatentPub. No. U.S. 2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods ofPreventing or Treating Inflammatory or Autoimmune Disorders byAdministering Integrin α_(v)β3 Antagonists in Combination With OtherProphylactic or Therapeutic Agents,” and International Publication No.WO 03/075741 A2, dated Sep. 18, 2003, entitled, “Methods of Preventingor Treating Disorders by Administering an Integrin αvβ3 Antagonist inCombination With an HMG-CoA Reductase Inhibitor or a Bisphosphonate,”each of which is incorporated herewith by reference in its entirety) toa subject in need thereof. In another specific embodiment, the inventionprovides methods of preventing, treating, managing, and/or amelioratinga fungal respiratory infection or one or more symptoms thereof, saidmethods comprising administering to a subject in need thereof aneffective amount of one or more antibodies of the invention incombination with an effective amount of siplizumab (MedImmune, Inc.,International Pub. No. WO 02/069904) to a subject in need thereof. Inanother embodiment, the invention provides methods of preventing,treating, managing, and/or ameliorating a fungal respiratory infectionor one or more symptoms thereof, said methods comprising administeringan effective amount of one or more antibodies of the invention incombination with an effective amount of one or more EphA2 inhibitors(e.g., one or more anti-EphA2 antibodies (MedImmune, Inc.; InternationalPublication No. WO 02/102974 A4, dated Dec. 27, 2002, entitled “MutantProteins, High Potency Inhibitory Antibodies and FIMCH CrystalStructure,” International Publication No. 03/094859 A2, dated Nov. 20,2003, entitled “EphA2 Monoclonal Antibodies and Methods of Use Thereof,”U.S. application Ser. No. 10/436,783; and U.S. Appn. No. 60/379,368,each of which is incorporated herewith by reference)) to a subject inneed thereof. In yet another embodiment, the invention provides methodsof preventing, treating, managing, and/or ameliorating a fungalinfection, preferably a fungal respiratory infection, or one or moresymptoms thereof, said methods comprising administering an effectiveamount of one or more antibodies of the invention in combination with aneffective amount of VITAXIN™, siplizumab, and/or EphA2 to a subject inneed thereof.

The invention encompasses methods for preventing the development offungal respiratory infections in a patient expected to suffer from afungal infection, preferably a fungal respiratory infection, or atincreased risk of such an infection. Such subjects include, but are notlimited to, patients with suppressed immune systems (e.g., patientsorgan-transplant recipients, AIDS patients, patients undergoingchemotherapy, patients with carcinoma of the esophagus with obstruction,patients with tracheobronchial fistula, patients with neurologicaldiseases (e.g., caused by stroke, amyotorphic lateral sclerosis,multiple sclerosis, and myopathies), and patients already suffering froma respiratory condition, particularly a respiratory infection). In aspecific embodiment, the patient suffers from bronchopulmonarydysplasia, congenital heart disease, cystic fibrosis, and/or acquired orcongenital immunodeficiency. In another specific embodiment, the patientis an infant born prematurely, an infant, a child, an elderly human, ora human in a group home, nursing home, or some other type ofinstitution. The invention also encompasses methods of preventing,managing, treating, and/or ameliorating a respiratory condition or oneor more symptoms thereof in patients who are susceptible to adversereactions to conventional therapies for respiratory conditions for whichno therapies are available.

The antibody formulations of the invention or combination therapies ofthe invention may be used as the first, second, third, fourth, or fifththerapy to prevent, manage, treat, and/or ameliorate a fungal infection,preferably a fungal respiratory infection or one or more symptomthereof. The invention also includes methods of preventing, treating,managing, and/or ameliorating a fungal infection, preferably a fungalrespiratory infection or one or more symptoms thereof in a patientundergoing therapies for other disease or disorders. The inventionencompasses methods of preventing, managing, treating, and/orameliorating a fungal infection, preferably a fungal respiratoryinfection or one or more symptoms thereof in a patient before anyadverse effects or intolerance to therapies other antibody formulationsof the invention develops. The invention also encompasses methods ofpreventing, treating, managing, and/or ameliorating a fungal infection,preferably a fungal respiratory infection or a symptom thereof inrefractory patients. In certain embodiments, a patient with a fungalinfection, preferably a fungal respiratory infection, is refractory to atherapy when the infection has not significantly been eradicated and/orthe symptoms have not been significantly alleviated. The determinationof whether a patient is refractory can be made either in vivo or invitro by any method known in the art for assaying the effectiveness of atreatment of infections, using art-accepted meanings of “refractory” insuch a context. In various embodiments, a patient with a fungalinfection, preferably a fungal respiratory infection, is refractory whenfungal replication has not decreased or has increased. The inventionalso encompasses methods of preventing the onset or reoccurrence offungal infections, preferably fungal respiratory infections, in patientsat risk of developing such infections. The invention also encompassesmethods of preventing, managing, treating, and/or ameliorating a fungalinfection, preferably a fungal respiratory infection, or a symptomthereof in patients who are susceptible to adverse reactions toconventional therapies. The invention further encompasses methods forpreventing, treating, managing, and/or ameliorating fungal infections,preferably fungal respiratory infections, for which no anti-fungaltherapy is available.

The invention encompasses methods for preventing, treating, managing,and/or ameliorating a fungal infection, preferably a fungal respiratoryinfection, or a symptom thereof in a patient who has proven refractoryto therapies other than antibody formulations of the invention but areno longer on these therapies. In certain embodiments, the patients beingmanaged or treated in accordance with the methods of this invention arepatients already being treated with antibiotics, anti-virals,anti-fungals, or other biological therapy/immunotherapy. Among thesepatients are refractory patients, patients who are too young forconventional therapies, and patients with reoccurring fungal infectionsdespite management or treatment with existing therapies.

The present invention provides methods for preventing, treating,managing, and/or ameliorating a fungal infection, preferably a fungalrespiratory infection, or one or more symptoms thereof as an alternativeto other conventional therapies. In specific embodiments, the patientbeing managed or treated in accordance with the methods of the inventionis refractory to other therapies or is susceptible to adverse reactionsfrom such therapies. The patient may be a person with a suppressedimmune system (e.g., post-operative patients, chemotherapy patients, andpatients with immunodeficiency disease), a person with impaired renal orliver function, the elderly, children, infants, infants bornprematurely, persons with neuropsychiatric disorders or those who takepsychotropic drugs, persons with histories of seizures, or persons onmedication that would negatively interact with conventional agents usedto prevent, manage, treat, and/or ameliorate a fungal infection,preferably a fungal respiratory infection, or one or more symptomsthereof.

Fungal infection therapies and their dosages, routes of administrationand recommended usage are known in the art and have been described insuch literature as the Physician's Desk Reference (59th ed., 2005).

5.6. Methods of Administering the Antibody Formulations

The invention provides methods of treatment, management, prophylaxis,and amelioration of a disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, autoimmune diseases, inflammatory diseases, proliferativediseases, or infections (preferably, respiratory infections), or one ormore symptoms thereof by administrating to a subject of an effectiveamount of liquid formulations of the invention. Various delivery systemsare known and can be used to administer a liquid formulation of thepresent invention or a prophylactic or therapeutic agent. Methods ofadministering antibody liquid formulations of the present invention or atherapy (e.g., a prophylactic or therapeutic agent) include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural administration,topical administration, and mucosal administration (e.g., intranasal andoral routes). In a specific embodiment, liquid formulations of thepresent invention are administered intramuscularly, intravenously, orsubcutaneously. In a preferred embodiment, the liquid formulations ofthe invention are administered subcutaneously. The formulations may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In a specific embodiment, theliquid formulations of the invention are administered intratumorally orat the site of inflammation.

In a specific embodiment, the antibody formulations of the inventioncomprise a pharmaceutically acceptable carrier. In a preferredembodiment, the pharmaceutically acceptable carrier is water forinjection, USP, 5% dextrose in water (D5W) or saline.

Generally, the antibodies (including antibody fragments thereof) thatimmunospecifically bind to an IL-9 polypeptide contained in the liquidformulations of the invention are derived from a subject that is of thesame species origin or species reactivity as recipient of the liquidformulations of the invention. Thus, in a preferred embodiment, liquidformulations of the invention comprising human or humanized antibodiesthat immunospecifically bind to an IL-9 polypeptide contained in theliquid formulations of the invention are administered to a human patientfor therapy or prophylaxis.

The invention also provides that a liquid formulation of the presentinvention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of antibody (includingantibody fragment thereof). Preferably, the liquid formulations of thepresent invention are in a hermetically sealed container indicating thequantity and concentration of the antibody (including antibody fragmentthereof). Preferably, the liquid formulation of the present invention issupplied in a hermetically sealed container and comprises at least 15mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, 250 mg/ml,or 300 mg/ml of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide, in a quantity of 1 ml,2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 mland, most preferably, 1.2 ml. In a specific embodiment of the invention,a liquid formulation of the invention is supplied in a hermeticallysealed container and comprises at least 15 mg/ml, at least 20 mg/ml, atleast 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150mg/ml, at least 175 mg/ml, at least 200 mg/ml, at least 250 mg/ml or atleast 300 mg/ml of an antibody (including antibody fragment thereof)that immunospecifically binds to an IL-9 polypeptide (e.g., 4D4, 4D4H2-1D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4 or an antigen-binding fragment thereof) forintravenous injections, and at least 15 mg/ml, 20 mg/ml, 50 mg/ml, 80mg/ml 100 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, 250 mg/ml or 300 mg/mlan antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide (e.g., 4D4, 4D4H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or a fragment thereof) for repeated subcutaneousadministration. In a specific embodiment, an antibody formulation of thepresent invention may be produced by lyophilizing the aqueous antibodyformulation. In a specific embodiment, the lyophilized antibody aqueousantibody solution may be reconstituted with a pharmaceuticallyacceptable carrier. In a specific embodiment, a pharmaceuticallyacceptable carrier is water for injection, USP, 5% dextrose in water(D5W) or saline.

The amount of a liquid formulation of the present invention which willbe effective in the treatment, management, prevention or amelioration ofa disease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereofcan be determined by standard clinical techniques well-known in the artor described herein. The precise dose to be employed in the formulationwill also depend on the route of administration, and the seriousness ofthe inflammatory disorder, autoimmune disorder or cancer, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

For formulations of the antibodies, proteins, polypeptides, peptides andfusion proteins encompassed by the invention, the dosage administered toa patient may be calculated using the patient's weight in kilograms (kg)multiplied by the dose to be administered in mg/kg. The required volume(in mL) to be given is then determined by taking the mg dose requireddivided by the concentration of the antibody formulation. The finalcalculated required volume will be obtained by pooling the contents ofas many vials as are necessary into syringe(s) to administer theantibody formulation of the invention. The final calculated requiredvolume will be obtained by pooling the contents of as many vials as arenecessary into syringe(s) to administer the drug. A maximum volume of2.0 mL of the antibody formulation can be injected per site. The dose(in mL) can be calculated using the following formula: Dose(mL)=[volunteer weight](kg)×[dose]mg/kg÷100 mg/mL of the antibodyformulation. Generally, human antibodies have a longer half-life withinthe human body than antibodies from other species due to the immuneresponse to the foreign polypeptides. Thus, lower dosages of humanantibodies and less frequent administration is often possible. Further,the dosage, volume and frequency of administration of liquidformulations of the present invention may be reduced by increasing theconcentration of an antibody (including antibody fragment thereof) inthe formulations, increasing affinity and/or avidity of the antibody(including antibody fragment thereof), and/or increasing the half-lifeof the antibody (including antibody fragment thereof).

In a specific embodiment, the dosage administered to a patient will becalculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg. The required volume (in mL) to begiven is then determined by taking the mg dose required divided by theconcentration of the antibody (including antibody fragment thereof) inthe formulations (100 mg/mL). The final calculated required volume willbe obtained by pooling the contents of as many vials as are necessaryinto syringe(s) to administer the drug. A maximum volume of 2.0 mL ofantibody (including antibody fragment thereof) in the formulations canbe injected per site.

Exemplary doses of a small molecule include milligram or microgramamounts of the small molecule per kilogram of subject or sample weight(e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram).

In a specific embodiment, 0.1 to 20 mg/kg/week, preferably 1 to 15mg/kg/week, more preferably 2 to 8 mg/week, even more preferably 3 to 7mg/kg/week, and most preferably 4 to 6 mg/kg/week of an antibody(including antibody fragment thereof) that immunospecifically binds toan IL-9 polypeptide (e.g., 4D4, 4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9,7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5, or 7F3com-3D4 or afragment thereof) in a liquid formulation of the invention isadministered to a subject with an inflammatory disorder, an autoimmunedisorder or cancer. In another embodiment, a subject is administered oneor more doses of a prophylactically or therapeutically effective amountof a liquid formulation of the invention, wherein the prophylacticallyor therapeutically effective amount is not the same for each dose.

In one embodiment, a liquid formulation of the invention is administeredin a dosing regimen that maintains the plasma concentration of theantibody immunospecific for α_(v)β₃ at a desirable level (e.g., about0.1 to about 100 μg/ml), which continuously blocks the an IL-9polypeptide activity. In a specific embodiment, the plasma concentrationof the antibody is maintained at 0.2 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml,3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml,15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml or50 μg/ml. The plasma concentration that is desirable in a subject willvary depending on several factors, including but not limited to, thenature of the disease or disorder, the severity of the disease ordisorder and the condition of the subject. Such dosing regimens areespecially beneficial in prevention, treatment, management andamelioration of a chronic disease or disorder.

In specific embodiments, a liquid formulation of the inventioncomprising a conjugated antibody (including antibody fragment thereof)immunospecific for an IL-9 polypeptide is administered intermittently.As used herein, “a conjugated antibody or antibody fragment” refers toan antibody (including antibody fragment thereof) that is conjugated orfused to another moiety, including but not limited to, a heterologouspeptide, polypeptide, another antibody (including antibody fragmentthereof), a marker sequence, a diagnostic agent, a therapeutic moiety, atherapeutic drug, a radioactive metal ion, a polymer, albumin, and asolid support.

In another embodiment, a subject, preferably a human, is administeredone or more doses of a prophylactically or therapeutically effectiveamount of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide (e.g., 4D4, 4D4H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or a fragment thereof) in a liquid formulation of theinvention, wherein the dose of a prophylactically or therapeuticallyeffective amount of the antibody (including antibody fragment thereof)in the liquid formulation of the invention administered to said subjectis increased by, e.g., 0.01 μg/kg, 0.02 μg/kg, 0.04 μg/kg, 0.05 gg/kg,0.06 μg/kg, 0.08 μg/kg, 0.1 μg/kg, 0.2 μg/kg, 0.25 μg/kg, 0.5 μg/kg,0.75 μg/kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 4 μg/kg, 5 μg/kg, 10 μg/kg, 15μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 pg/kg, 75 μg/kg, 80 μg/kg, 85μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, or 125 μg/kg, as treatmentprogresses.

In another embodiment, a subject, preferably a human, is administeredone or more doses of a prophylactically or therapeutically effectiveamount of an antibody (including antibody fragment thereof) thatimmunospecifically binds to an IL-9 polypeptide (e.g., 4D4, 4D4H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or a fragment thereof) in a liquid formulation of theinvention, wherein the dose of a prophylactically or therapeuticallyeffective amount of the antibody (including antibody fragment thereof)in the liquid formulation of the invention administered to said subjectis decreased by, e.g., 0.01 μg/kg, 0.02 μg/kg, 0.04 μg/kg, 0.05 μg/kg,0.06 μg/kg, 0.08 μg/kg, 0.1 μg/kg, 0.2 μg/kg, 0.25 μg/kg, 0.5 μg/kg,0.75 μg/kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 4 μg/kg, 5 μg/kg, 10 μg/kg, 15μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80 μg/kg, 85μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, or 125 μg/kg, as treatmentprogresses.

The dosages of prophylactic or therapeutically agents are described inthe Physicians' Desk Reference (56th ed., 2002 and 57th ed., 2003).

5.7. Biological Assays

The antibodies (including antibody fragment thereof) of the liquidformulations of the invention may be characterized in a variety of wayswell-known to one of skill in the art. In particular, antibodies(including antibody fragments thereof) of the liquid formulations of theinvention may be assayed for the ability to immunospecifically bind toan IL-9 polypeptide. Such an assay may be performed in solution (e.g.,Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991, Nature354:82-84), on chips (Fodor, 1993, Nature 364:555-556), on bacteria(U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), on plasmids (Cull et al., 1992, Proc. Natl.Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science249:386-390; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310) (each ofthese references is incorporated herein in its entirety by reference).Antibodies (including antibody fragments thereof) that have beenidentified to immunospecifically bind to an IL-9 polypeptide can then beassayed for their specificity and affinity for an IL-9 polypeptide.

The antibodies (including antibody fragments thereof) of the liquidformulations of the invention may be assayed for immunospecific bindingto an IL-9 polypeptide and cross-reactivity with other antigens by anymethod known in the art. Immunoassays which can be used to analyzeimmunospecific binding and cross-reactivity include, but are not limitedto, competitive and non-competitive assay systems using techniques suchas western blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety). Exemplary immunoassays are described brieflybelow (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1 to 4 hours) at 40° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 40° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, incubating the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), incubating themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, incubating themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. In a preferred embodiment, an ELISA may be performedby coating a high binding 96-well microtiter plate (Costar) with 2 μg/mlof rhu-IL-9 in PBS overnight. Following three washes with PBS, the plateis incubated with three-fold serial dilutions of Fab at 25° C. for 1hour. Following another three washes of PBS, 1 μg/ml anti-humankappa-alkaline phosphatase-conjugate is added and the plate is incubatedfor 1 hour at 25° C. Following three washes with PBST, the alkalinephosphatase activity is determined in 50 μl/AMP/PPMP substrate. Thereactions are stopped and the absorbance at 560 nm is determined with aVMAX microplate reader. For further discussion regarding ELISAs see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of the contained in a liquid formulation ofthe present invention or a fragment thereof for an IL-9 polypeptide andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, an IL-9 polypeptide is incubatedwith an antibody of the present invention conjugated to a labeledcompound (e.g., ³H or ¹²⁵I) in the presence of increasing amounts of anunlabeled second antibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies of the liquid formulations ofthe invention to an IL-9 polypeptide. BIAcore kinetic analysis comprisesanalyzing the binding and dissociation of an IL-9 polypeptide from chipswith immobilized antibodies of the invention on their surface. A typicalBIAcore kinetic study involves the injection of 250 μL of an antibodyreagent (mab, Fab) at varying concentration in HBS buffer containing0.005% Tween-20 over a sensor chip surface, onto which has beenimmobilized the antigen. The flow rate is maintained constant at 75μL/min. Dissociation data is collected for 15 min. or longer asnecessary. Following each injection/dissociation cycle, the bound mAb isremoved from the antigen surface using brief, 1 min. pulses of diluteacid, typically 10-100 mM HCl, though other regenerants are employed asthe circumstances warrant. More specifically, for measurement of therates of association, k_(on), and dissociation, k_(off), the antigen isdirectly immobilized onto the sensor chip surface through the use ofstandard amine coupling chemistries, namely the EDCINHS method(EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a 5-100 nM solutionof the antigen in 10 mM NaOAc, pH4 or pH5 is prepared and passed overthe EDC/NHS-activated surface until approximately 30-50 RU's worth ofantigen are immobilized. Following this, the unreacted active esters are“capped” off with an injection of 1M Et-NH2. A blank surface, containingno antigen, is prepared under identical immobilization conditions forreference purposes. Once an appropriate surface has been prepared, asuitable dilution series of each one of the antibody reagents isprepared in HBS/Tween-20, and passed over both the antigen and referencecell surfaces, which are connected in series. The range of antibodyconcentrations that are prepared varies, depending on what theequilibrium binding constant, K_(D), is estimated to be. As describedabove, the bound antibody is removed after each injection/dissociationcycle using an appropriate regenerant.

The antibodies (including antibody fragments thereof) of the liquidformulations of the invention can also be assayed for their ability toinhibit the binding of IL-9 to its host cell receptor using techniquesknown to those of skill in the art. For example, cells expressing IL-9receptor can be contacted with IL-9 in the presence or absence of anantibody (including antibody fragment thereof) of the liquidformulations of the invention and the ability of the antibody (includingantibody fragment thereof) to inhibit IL-9's binding can measured by,for example, flow cytometry or a scintillation assay. IL-9 or theantibody (including antibody fragment thereof) contained in the liquidformulation of the invention can be labeled with a detectable compoundsuch as a radioactive label (e.g., 32P, 35S, and 125I) or a fluorescentlabel (e.g., fluorescein isothiocyanate, rhodanine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) toenable detection of an interaction between IL-9 and its host cellreceptor. Alternatively, the ability of antibodies (including antibodyfragment thereof) of the liquid formulations of the invention to inhibitIL-9 from binding to its receptor can be determined in cell-free assays.For example, an IL-9 polypeptide can be contacted with an antibody(including antibody fragment thereof) of the liquid formulations of theinvention and the ability of the antibody (including antibody fragmentthereof) to inhibit the IL-9 polypeptide from binding to its host cellreceptor can be determined. Preferably, the antibody (including antibodyfragment thereof) of the liquid formulations of the invention of theinvention is immobilized on a solid support and an IL-9 polypeptide islabeled with a detectable compound. Alternatively, an IL-9 polypeptideis immobilized on a solid support and the antibody (including antibodyfragment thereof) contained within a liquid formulation of the inventionis labeled with a detectable compound. An IL-9 may be partially orcompletely purified (e.g., partially or completely free of otherpolypeptides) or part of a cell lysate. Further, an IL-9 polypeptide maybe a fusion protein comprising IL-9, a derivative, analog or fragmentthereof and a domain such as glutathionine-5-transferase. Alternatively,an IL-9 polypeptide can be biotinylated using techniques well known tothose of skill in the art (e.g., biotinylation kit, Pierce Chemicals;Rockford, Ill.).

In a specific embodiment, the ability of antibodies (including antibodyfragments thereof) of the liquid formulations of the invention toinhibit IL-9 from binding to its host cell receptor can be measured bycell proliferation assays. In a preferred embodiment, the murine TS1-RA3T cell line expressing both human and murine IL-9Rα may be growncontinuously in growth medium (DMEM) containing rhuIL-9 (25 ng/ml, R & DSystems). Upon withdrawal of rhuIL-9, TS 1-RA3 undergoes cell death in18-24 hours. TS1-RA3 cells grown in RPMI 1640 (ATCC) medium supplementedwith 10% FBS and 25 ng/ml rHu-IL9. Prior to the assay, the cells arewashed with media containing no IL-9 and resuspended at 5×10⁵ cells/mlin media containing 2 ng/ml rhuIL-9. The cells are distributed into ablack clear bottom non-binding 96-well microtiter plate (100 μlcells/well) and 100 ml of serially diluted variant Fabs is then added tothe plate. The plate is incubated at 72 hours at 37° C., 5% CO2. 20μl/well of Alamar blue® is added, and the cells are incubated for anadditional 4-5 hours. Cell metabolism is quantitated using a fluorimeterwith excitation at 555 nm and emission at 590 nm.

5.7.1. In Vitro Studies

The antibodies, compositions, or combination therapies of the inventioncan be tested in vitro and/or in vivo for their ability to modulate thebiological activity of immune cells (e.g., T cells, neutrophils, andmast cells), endothelial cells, and epithelial cells. The ability of anantibody, composition, or combination therapy of the invention tomodulate the biological activity of immune cells (e.g., T cells, Bcells, mast cells, macrophages, neutrophils, and eosinophils),endothelial cells, and epithelial cells can be assessed by: detectingthe expression of antigens (e.g., activation of genes by IL-9, such as,but not limited to, mucin genes (e.g., MUC2, MUC5AC, MUC5B, and MUC6)and genes involved in lymphocyte activation (e.g., Lgamma-6A/E));detecting the proliferation of immune cells, endothelia cells and/orepithelial cells; detecting the activation of signaling molecules (e.g.,the phosphorylation of Stat2, the phosphorylation of JAK3, or thephosphorylation of the IL-9R); detecting the effector function of immunecells (e.g., T cells, B cells, mast cells, macrophages, neutrophils, andeosinophils), endothelial cells, and/or epithelial cells; or detectingthe differentiation of immune cells, endothelial cells, and/orepithelial cells. Techniques known to those of skill in the art can beused for measuring these activities. For example, cellular proliferationcan be assayed by 3H-thymidine incorporation assays and trypan blue cellcounts. Antigen expression can be assayed, for example, by immunoassaysincluding, but are not limited to, competitive and non-competitive assaysystems using techniques such as western blots, immunohistochemistryradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, and FACS analysis. Theactivation of signaling molecules can be assayed, for example, by kinaseassays and electrophoretic shift assays (EMSAs). Mast cell degranulationcan be assayed, for example by measuring serotonin (5-HT) release orhistamine release with high-performance liquid chromatogoraphy (see,e.g., Taylor et al. 1995 Immunology 86(3): 427-433 and Kurosawa et al.,1998 Clin Exp Allergy 28(8): 1007-1012).

The antibodies, compositions, or combination therapies of the inventionare preferably tested in vitro and then in vivo for the desiredtherapeutic or prophylactic activity prior to use in humans. Forexample, assays which can be used to determine whether administration ofa specific pharmaceutical composition is indicated include cell cultureassays in which a patient tissue sample is grown in culture and exposedto, or otherwise contacted with, a pharmaceutical composition, and theeffect of such composition upon the tissue sample is observed. Thetissue sample can be obtained by biopsy from the patient. This testallows the identification of the therapeutically most effective therapy(e.g., prophylactic or therapeutic agent) for each individual patient.In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved a disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9R or oneor more subunits thereof, an inflammatory disorder, an autoimmunedisorder, a proliferative disorder, or an infection (preferably, arespiratory infection) to determine if a pharmaceutical composition ofthe invention has a desired effect upon such cell types.

The effect of an antibody, a composition, or a combination therapy ofthe invention on peripheral blood lymphocyte counts can bemonitored/assessed using standard techniques known to one of skill inthe art. Peripheral blood lymphocytes counts in a subject can bedetermined by, e.g., obtaining a sample of peripheral blood from saidsubject, separating the lymphocytes from other components of peripheralblood such as plasma using, e.g., Ficoll-Hypaque (Pharmacia) gradientcentrifugation, and counting the lymphocytes using trypan blue.Peripheral blood T-cell counts in subject can be determined by, e.g.,separating the lymphocytes from other components of peripheral bloodsuch as plasma using, e.g., a use of Ficoll-Hypaque (Pharmacia) gradientcentrifugation, labeling the T-cells with an antibody directed to aT-cell antigen which is conjugated to FITC or phycoerythrin, andmeasuring the number of T-cells by FACS.

The methods of the invention for treating, managing, preventing, and/orameliorating a viral respiratory infection or one or more symptomsthereof can be tested for their ability to inhibit viral replication orreduce viral load in in vitro assays. For example, viral replication canbe assayed by a plaque assay such as described, e.g., by Johnson et al.,1997, Journal of Infectious Diseases 176:1215-1224 176:1215-1224. Theantibodies, compositions, or combination therapies administeredaccording to the methods of the invention can also be assayed for theirability to inhibit or downregulate the expression of viral polypeptides.Techniques known to those of skill in the art, including, but notlimited to, western blot analysis, northern blot analysis, and RT-PCRcan be used to measure the expression of viral polypeptides.

The methods of the invention for preventing, treating, managing, and/orameliorating a respiratory infection or one or more symptoms thereof canbe tested for activity against bacteria causing respiratory infectionsin in vitro assays well-known in the art. In vitro assays known in theart can also be used to test the existence or development of resistanceof bacteria to a therapy (e.g., an antibody of the invention, otherprophylactic or therapeutic agent, a combination thereof, or acomposition thereof) of the invention. Such in vitro assays aredescribed in Gales et al., 2002, Diag. Nicrobiol. Infect. Dis.44(3):301-311; Hicks et al., 2002, Clin. Microbiol. Infect. 8(11):753-757; and Nicholson et al., 2002, Diagn. Microbiol. Infect. Dis.44(1): 101-107.

The therapies (e.g., an antibody of the liquid formulations of theinvention alone or in combination with prophylactic or therapeuticagents, other than antibodies of the invention) of the invention fortreating, managing, preventing, and/or ameliorating a respiratoryinfection or one or more symptoms thereof can be tested for anti-fungalactivity against different species of fungus. Any of the standardanti-fungal assays well-known in the art can be used to assess theanti-fungal activity of a therapy. The anti-fungal effect on differentspecies of fungus can be tested. The tests recommended by the NationalCommittee for Clinical Laboratories (NCCLS) (See National Committee forClinical Laboratories Standards. 1995, Proposed Standard M27T.Villanova, Pa., all of which is incorporated herein by reference in itsentirety) and other methods known to those skilled in the art (Pfalleret al., 1993, Infectious Dis. Clin. N. Am. 7: 435-444) can be used toassess the anti-fungal effect of a therapy. The antifungal properties ofa therapy may also be determined from a fungal lysis assay, as well asby other methods, including, inter alia, growth inhibition assays,fluorescence-based fungal viability assays, flow cytometry analyses, andother standard assays known to those skilled in the art.

For example, the anti-fungal activity of a therapy can be tested usingmacrodilution methods and/or microdilution methods using protocolswell-known to those skilled in the art (see, e.g., Clancy et al., 1997Journal of Clinical Microbiology, 35(11): 2878-82; Ryder et al., 1998,Antimicrobial Agents and Chemotherapy, 42(5): 1057-61; U.S. Pat. No.5,521,153; U.S. Pat. No. 5,883,120, U.S. Pat. No. 5,521,169, all ofwhich are incorporated by reference in their entirety). Briefly, afungal strain is cultured in an appropriate liquid media, and grown atan appropriate temperature, depending on the particular fungal strainused for a determined amount of time, which is also depends on theparticular fungal strain used. An innoculum is then preparedphotometrically and the turbidity of the suspension is matched to thatof a standard, e.g., a McFarland standard. The effect of a therapy onthe turbidity of the inoculum is determined visually orspectrophotometrically. The minimal inhibitory concentration (“MIC”) ofthe therapy is determined, which is defined as the lowest concentrationof the lead compound which prevents visible growth of an inoculum asmeasured by determining the culture turbidity.

The anti-fungal activity of a therapy can also be determined utilizingcolorimetric based assays well-known to one of skill in the art. Oneexemplary calorimetric assay that can be used to assess the anti-fungalactivity of a therapy is described by Pfaller et al. (1994, Journal ofClinical Microbiology, 32(8): 1993-6, which is incorporated herein byreference in its entirety; also see Tiballi et al., 1995, Journal ofClinical Microbiology, 33(4): 915-7). This assay employs a colorimetricendpoint using an oxidation-reduction indicator (Alamar Biosciences,Inc., Sacramento Calif.).

The anti-fungal activity of a therapy can also be determined utilizingphotometric assays well-known to one of skill in the art (see, e.g.,Clancy et al., 1997 Journal of Clinical Microbiology, 35(11): 2878-82;Jahn et al., 1995, Journal of Clinical Microbiology, 33(3): 661-667,each of which is incorporated herein by reference in its entirety). Thisphotometric assay is based on quantifying mitochondrial respiration byviable fungi through the reduction of3-(4,5-dimethyl-2thiazolyl)-2,5,-diphenyl-2H-tetrazolium bromide (MTT)to formazan. MIC's determined by this assay are defined as the highestconcentration of the test therapy associated with the first precipitousdrop in optical density. In some embodiments, the therapy is assayed foranti-fungal activity using macrodilution, microdilution and MTT assaysin parallel.

Further, any in vitro assays known to those skilled in the art can beused to evaluate the prophylactic and/or therapeutic utility of anantibody, a composition, a combination therapy disclosed herein for arespiratory infection or one or more symptoms thereof.

5.7.2. In Vivo Assays

The antibodies, compositions, or combination therapies of the inventioncan be tested in suitable animal model systems prior to use in humans.Such animal model systems include, but are not limited to, rats, mice,chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal systemwell-known in the art may be used. Several aspects of the procedure mayvary; said aspects include, but are not limited to, the temporal regimeof administering the therapies (e.g., prophylactic and/or therapeuticagents), whether such therapies are administered separately or as anadmixture, and the frequency of administration of the therapies.

Animal models for autoimmune disorders can also be used to assess theefficacy of an antibody, a composition, or a combination therapy of theinvention. Animal models for autoimmune disorders such as type 1diabetes, thyroid autoimmunity, systemic lupus erythematosus, andglomerulonephritis have been developed (Flanders et al., 1999,Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515;Foster, 1999, Semin. Nephrol. 19:12-24).

Efficacy in preventing, treating, managing, and/or ameliorating anautoimmune disorder may be demonstrated, e.g., by detecting the abilityof an antibody, a composition, or a combination therapy of the inventionto reduce one or more symptoms of the autoimmune disorder, to reducemean absolute lymphocyte counts, to decrease T cell activation, todecrease T cell proliferation, to reduce cytokine production, or tomodulate one or more particular cytokine profiles. Efficacy inpreventing or treating psoriasis may be demonstrated, e.g., by detectingthe ability of an antibody, fragment thereof, or composition of theinvention to reduce one or more symptoms of psoriasis, to reduce meanabsolute lymphocyte counts, to reduce cytokine production, to modulateone or more particular cytokine profiles, to decrease scaling, todecrease erythema, to decrease plaque elevation, to decrease T cellactivation in the dermis or epidermis of an affected area, to decrease Tcell infiltration to the dermis or epidermis of an affected area, toreduce PASI, to improve the physician's global assessment score, or toimprove quality of life.

Animal models for cancer can be used to assess the efficacy of anantibody, a composition, or a combination therapy of the invention.Examples of animal models for lung cancer include, but are not limitedto, lung cancer animal models described by Zhang & Roth (1994, In Vivo8(5):755-69) and a transgenic mouse model with disrupted p53 function(see, e.g., Morris et al., 1998, J La State Med Soc 150(4):179-85). Anexample of an animal model for breast cancer includes, but is notlimited to, a transgenic mouse that overexpresses cyclin D1 (see, e.g.,Hosokawa et al., 2001, Transgenic Res 10(5):471-8). An example of ananimal model for colon cancer includes, but is not limited to, a TCR band p53 double knockout mouse (see, e.g., Kado et al., 2001, Cancer Res61(6):2395-8). Examples of animal models for pancreatic cancer include,but are not limited to, a metastatic model of Panc02 murine pancreaticadenocarcinoma (see, e.g., Wang et al., 2001, Int J Pancreatol29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumours(see, e.g., Ghaneh et al., 2001, Gene Ther 8(3):199-208). Examples ofanimal models for non-Hodgkin's lymphoma include, but are not limitedto, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryantet al., 2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenicmouse (see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA95(23):13853-8). An example of an animal model for esophageal cancerincludes, but is not limited to, a mouse transgenic for the humanpapillomavirus type 16 E7 oncogene (see, e.g., Herber et al., 1996, JVirol 70(3):1873-81). Examples of animal models for colorectalcarcinomas include, but are not limited to, Apc mouse models (see, e.g.,Fodde & Smits, 2001, Trends Mol Med 7(8):369-73 and Kuraguchi et al.,2000, Oncogene 19(50):5755-63).

The anti-inflammatory activity of an antibody, a composition, or acombination therapy of the invention can be determined by using variousexperimental animal models of inflammatory arthritis known in the artand described in Crofford L. J. and Wilder R. L., “Arthritis andAutoimmunity in Animals,” in Arthritis and Allied Conditions: A Textbookof Rheumatology, McCarty (eds.), Chapter 30 (Lee and Febiger, 1993).Experimental and spontaneous animal models of inflammatory arthritis andautoimmune rheumatic diseases can also be used to assess theanti-inflammatory activity of the antibodies, compositions, orcombination therapies of invention.

The anti-inflammatory activity of an antibody, a composition, or acombination therapy of invention can also be assessed by measuring theinhibition of carrageenan-induced paw edema in the rat, using amodification of the method described in Winter C. A. et al.,“Carrageenan-Induced Edema in Hind Paw of the Rat as an Assay forAnti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111, 544-547, (1962).This assay has been used as a primary in vivo screen for theanti-inflammatory activity of most NSAIDs, and is considered predictiveof human efficacy. The anti-inflammatory activity of the test therapies(e.g., prophylactic or therapeutic agents) is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

In a specific embodiment of the invention where the experimental animalmodel used is adjuvant-induced arthritis rat model, body weight can bemeasured relative to a control group to determine the anti-inflammatoryactivity of an antibody, a composition, a combination therapy of theinvention.

Animal models for allergies and asthma are known in the art, such asconstant-flow inflation with end-inspiratory occlusion described inEwart et al., 1995 J Appi Physiol 79(2):560-566 and other assaysdescribed in, e.g., Komai et al., 2003 Br J Pharmacol 138(5): 912-920;Kenyon et al., 2003 Toxicol Appl Pharmacol 186(2): 90-100; Path et al.,2002 Am J Resp & Critical Care Med 166(6): 818-826; Martins et al., 1990Crit Care Med 19:515-519; Nicolaides et al., 1997 Proc Natl Acad Sci USA94:13175-13180; McLane et al., 1998 19:713-720; and Temann et al., 1998J Exp Med 188(7): 1307-1320. For example, the murine adoptive transfermodel is an animal model used to assess the efficacy an antibody, acomposition, or a combination therapy of the invention for theprevention, treatment, management, and/or asthma include. In the murineadoptive transfer model, aeroallergen provocation of TH1 or TH2recipient mice results in TH effector cell migration to the airways andis associated with an intense neutrophilic (TH1) and eosinophilic (TH2)lung mucosal inflammatory response (Cohn et al., 1997, J. Exp. Med.1861737-1747). Airway hypersensitivity can be induced in mice byovalbumin (Tomkinson et al., 2001, J. Immunol. 166:5792-5800) orSchistosoma mansoni egg antigen (Tesciuba et al., 2001, J. Immunol.167:1996-2003).

Efficacy in preventing or treating an inflammatory disorder may bedemonstrated, e.g., by detecting the ability of an antibody, acomposition, or a combination therapy of the invention to reduce one ormore symptoms of the inflammatory disorder, to decrease T cellactivation, to decrease T cell proliferation, to modulate one or morecytokine profiles, to reduce cytokine production, to reduce inflammationof a joint, organ or tissue or to improve quality of life.

Changes in inflammatory disease activity may also be assessed throughtender and swollen joint counts, patient and physician global scores forpain and disease activity, and the ESR/CRP. Progression of structuraljoint damage may be assessed by quantitative scoring of X-rays of hands,wrists, and feet (Sharp method). Changes in functional status in humanswith inflammatory disorders may be evaluated using the Health AssessmentQuestionnaire (HAQ), and quality of life changes are assessed with theSF.

The efficacy of an antibody, a composition, or a combination therapy ofthe invention in preventing, treating, managing, and/or amelioratingType I allergic reaction may be assessed by its ability to induceanti-IgE antibodies that inhibit IgE from binding to is receptor on mastcells or basophils in vitro. IgE levels can be assayed by immunoassays,gel electrophoresis followed by visualization, radioimmunosorbent test(RIST), radioallergosorbent test (RAST), or any other method known tothose skilled in the art.

Animal models for viral infections can also be used to assess theefficacy of an antibody, a composition, or a combination therapy of theinvention. Animal models for viral infections such as EBV-associateddiseases, gammaherpesviruses, infectious mononucleosis, simianimmunodeficiency virus (“SIV”), Borna disease virus infection,hepatitis, varicella virus infection, viral pneumonitis, Epstein-Barrvirus pathogenesis, feline immunodeficiency virus (“FIV”), HTLV type 1infection, human rotaviruses, and genital herpes have been developed(see, e.g., Hayashi et al., 2002, Histol Histopathol 17(4):1293-310;Arico et al., 2002, J Interferon Cytokine Res 22(11):1081-8; Flano etal., 2002, Immunol Res 25(3):201-17; Sauermann, 2001, Curr Mol Med1(4):515-22; Pletnikov et al., 2002, Front Biosci 7:d593-607; Engler etal., 2001, Mol Immunol 38(6):457-65; White et al., 2001, Brain Pathol11(4):475-9; Davis & Matalon, 2001, News Physiol Sci 16:185-90; Wang,2001, Curr Top Microbiol Immunol. 258:201-19; Phillips et al., 2000, JPsychopharmacol. 14(3):244-50; Kazanji, 2000, AIDS Res Hum Retroviruses.16(16):1741-6; Saif et al., 1996, Arch Virol Suppl. 12:153-61; andHsiung et al., 1984, Rev Infect Dis. 6(1):33-50).

Animal models for viral respiratory infections such as, but not limitedto, PIV (see, e.g., Shephard et al., 2003 Res Vet Sci 74(2): 187-190;Ottolini et al., 2002 J Infect Dis 186(12): 1713-1717), RSV (see, e.g.,Culley et al., 2002 J Exp Med 196(10): 1381-1386; and Curtis et al.,2002 Exp Biol Med 227(9): 799-802). In a specific embodiment, cottonrats are administered an antibody of the invention, a composition, or acombination therapy according to the methods of the invention,challenged with 10⁵ pfu of RSV, and four or more days later the rats aresacrificed and RSV titer and anti-RSV antibody serum titer isdetermined. Accordingly, a dosage that results in a 2 log decrease or a99% reduction in RSV titer in the cotton rat challenged with 10⁵ pfu ofRSV relative to the cotton rat challenged with 10⁵ pfu of RSV but notadministered the formulation is the dosage of the formulation that canbe administered to a human for the treatment, prevention or ameliorationof one or more symptoms associated with RSV infection. Further, inaccordance with this embodiment, the tissues (e.g., the lung tissues)from the sacrificed rats can be examined for histological changes.

The antibodies, compositions, or combination therapies of the inventioncan be tested for their ability to decrease the time course of viralinfection. The antibodies, compositions, or combination therapies of theinvention can also be tested for their ability to increase the survivalperiod of humans suffering from a viral infection by at least 25%,preferably at least 50%, at least 60%, at least 75%, at least 85%, atleast 95%, or at least 99%. Further, antibodies, compositions, orcombination therapies of the invention can be tested for their abilityreduce the hospitalization period of humans suffering from viralinfection by at least 60%, preferably at least 75%, at least 85%, atleast 95%, or at least 99%. Techniques known to those of skill in theart can be used to analyze the function of the antibodies, compositions,or combination therapies of the invention in vivo.

Animal models for bacterial infections can also be used to assess theefficacy of an antibody, a composition, or a combination therapy of theinvention. Animal models for bacterial infections such as H.pylori-infection, genital mycoplasmosis, primary sclerosing cholangitis,cholera, chronic lung infection with Pseudomonas aeruginosa,Legionnaires' disease, gastroduodenal ulcer disease, bacterialmeningitis, gastric Helicobacter infection, pneumococcal otitis media,experimental allergic neuritis, leprous neuropathy, mycobacterialinfection, endocarditis, Aeromonas-associated enteritis, Bacteroidesfragilis infection, syphilis, streptococcal endocarditis, acutehematogenous osteomyelitis, human scrub typhus, toxic shock syndrome,anaerobic infections, Escherichia coli infections, and Mycoplasmapneumoniae infections have been developed (see, e.g., Sugiyama et al.,2002, J Gastroenterol. 37 Suppl 13:6-9; Brown et al., 2001, Am J ReprodImmunol. 46(3):232-41; Vierling, 2001, Best Pract Res ClinGastroenterol. 15(4):591-610; Klose, 2000, Trends Microbiol.8(4):189-91; Stotland et al., 2000, Pediatr Pulmonol. 30(5):413-24;Brieland et al., 2000, Immunopharmacology 48(3):249-52; Lee, 2000,Baillieres Best Pract Res Clin Gastroenterol. 14(1):75-96; Koedel &Pfister, 1999, Infect Dis Clin North Am. 13(3):549-77; Nedrud, 1999,FEMS Immunol Med Microbiol. 24(2):243-50; Prellner et al., 1999, MicrobDrug Resist. 5(1):73-82; Vriesendorp, 1997, J Infect Dis. 176 Suppl2:S164-8; Shetty & Antia, 1996, Indian J Lepr. 68(1):95-104;Balasubramanian et al., 1994, Immunobiology 191(4-5):395-401; Carbon etal., 1994, Int J Biomed Comput. 36(1-2):59-67; Haberberger et al., 1991,Experientia. 47(5):426-9; Onderdonk et al., 1990, Rev Infect Dis. 12Suppl 2:S169-77; Wicher & Wicher, 1989, Crit Rev Microbiol.16(3):181-234; Scheld, 1987, J Antimicrob Chemother. 20 Suppl A:71-85;Emslie & Nade, 1986, Rev Infect Dis. 8(6):841-9; Ridgway et al., 1986,Lab Anim Sci. 36(5):481-5; Quimby & Nguyen, 1985, Crit Rev Microbiol.12(1):1-44; Onderdonk et al., 1979, Rev Infect Dis. 1(2):291-301; Smith,1976, Ciba Found Symp. (42):45-72, and Taylor-Robinson, 1976, Infection.4(1 Suppl):4-8).

The antibodies, compositions, or combination therapies of the inventioncan be tested for their ability to decrease the time course of bacterialinfection, preferably bacterial respiratory infection by at least 25%,preferably at least 50%, at least 60%, at least 75%, at least 85%, atleast 95%, or at least 99%. The antibodies, compositions, or combinationtherapies of the invention can also be tested for their ability toincrease the survival period of humans suffering from a bacterialinfection by at least 25%, preferably at least 50%, at least 60%, atleast 75%, at least 85%, at least 95%, or at least 99%. Further, theantibodies, compositions, or combination therapies administeredaccording to the methods of the invention can be tested for theirability reduce the hospitalization period of humans suffering frombacterial infection, preferably a bacterial respiratory infection, by atleast 60%, preferably at least 75%, at least 85%, at least 95%, or atleast 99%. Techniques known to those of skill in the art can be used toanalyze the function of the Antibodies of the invention, compositions,or combination therapies of the invention in vivo.

The efficacy of the antibodies, compositions, or combination therapiesof the invention for the prevention, management, treatment, oramelioration of a fungal infection can be assessed in animal models forsuch infections. Animal models for fungal infections such as Candidainfections, zygomycosis, Candida mastitis, progressive disseminatedtrichosporonosis with latent trichosporonemia, disseminated candidiasis,pulmonary paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystiscarinii pneumonia, cryptococcal meningitis, coccidioidalmeningoencephalitis and cerebrospinal vasculitis, Aspergillus nigerinfection, Fusarium keratitis, paranasal sinus mycoses, Aspergillusfumigatus endocarditis, tibial dyschondroplasia, Candida glabratavaginitis, oropharyngeal candidiasis, X-linked chronic granulomatousdisease, tinea pedis, cutaneous candidiasis, mycotic placentitis,disseminated trichosporonosis, allergic bronchopulmonary aspergillosis,mycotic keratitis, Cryptococcus neoformans infection, fungalperitonitis, Curvularia geniculata infection, staphylococcalendophthalmitis, sporotrichosis, and dermatophytosis have been developed(see, e.g., Arendrup et al., 2002, Infection 30(5):286-91; Kamei, 2001,Mycopathologia 152(1):5-13; Guhad et al., 2000, FEMS Microbiol Lett.192(1):27-31; Yamagata et al., 2000, J Clin Microbiol. 38(9):32606;Andrutis et al., 2000, J Clin Microbiol. 38(6):2317-23; Cock et al.,2000, Rev Inst Med Trop Sao Paulo 42(2):59-66; Shibuya et al., 1999,Microb Pathog. 27(3):123-31; Beers et al., 1999, J Lab Clin Med.133(5):423-33; Najvar et al., 1999, Antimicrob AgentsChemother.43(2):413-4; Williams et al., 1988, J Infect Dis.178(4):1217-21; Yoshida, 1988, Kansenshogaku Zasshi. 1998June;72(6):621-30; Alexandrakis et al., 1998, Br J Ophthalmol.82(3):306-11; Chakrabarti et al., 1997, J Med Vet Mycol. 35(4):295-7;Martin et al., 1997, Antimicrob Agents Chemother. 41(1):13-6; Chu etal., 1996, Avian Dis. 40(3):715-9; Fidel et al., 1996, J Infect Dis.173(2):425-31; Cole et al., 1995, FEMS Microbiol Lett. 15;126(2):177-80;Pollock et al., 1995, Nat Genet. 9(2):202-9; Uchida et al., 1994, Jpn J.Antibiot. 47(10):1407-12;: Maebashi et al., 1994, J Med Vet Mycol.32(5):349-59; Jensen & Schonheyder, 1993, J Exp Anim Sci. 35(4):155-60;Gokaslan & Anaissie, 1992, Infect Immun. 60(8):3339-44; Kurup et al.,1992, J. Immunol. 148(12):3783-8; Singh et al., 1990, Mycopathologia.112(3): 127-37; Salkowski & Balish, 1990, Infect Immun. 58(10):3300-6;Ahmad et al., 1986, Am J Kidney Dis. 7(2):153-6; Alture-Werber E, EdbergS C, 1985, Mycopathologia. 89(2):69-73; Kane et al., 1981, AntimicrobAgents Chemother. 20(5):595-9; Barbee et al., 1977, Am J Pathol.86(1):281-4; and Maestrone et al., 1973, Am J Vet Res. 34(6):833-6).Animal models for fungal respiratory infections such as Candidaalbicans, Aspergillus fumigatus, invasive pulmonary aspergillosis,Pneumocystis carinii, pulmonary cryptococcosis, Pseudomonas aeruginosa,Cunninghamella bertholletia (see, e.g., Aratani et al., 2002 Med Mycol40(6):557-563; Bozza et al., 2002 Microbes Infect 4(13): 1281-1290;Kurup et al., 2002 Int Arch Allergy Immunol 129(2):129-137; Hori et al.,2002 Eur J Immuno 32(5): 1282-1291; Rivera et al., 2002 J Immuno 168(7):3419-3427; Vassallo et al., 2001, Am J Respir Cell Mol Biol 25(2):203-211; Wilder et al., 2002 Am J Respir Cell Mol Biol 26(3): 304-314;Yonezawa et al., 2000 J Infect Chemother 6(3): 155-161; Cacciapuoti etal., 2000 Antimicrob Agents Chemother 44(8): 2017-2022; and Honda etal., 1998 Mycopathologia 144(3):141-146).

The antibodies, compositions, or combination therapies of the inventioncan be tested for their ability to decrease the time course of fungalrespiratory infection by at least 25%, preferably at least 50%, at least60%, at least 75%, at least 85%, at least 95%, or at least 99%. Theantibodies, compositions, or combination therapies of the invention canalso be tested for their ability to increase the survival period ofhumans suffering from a fungal respiratory infection by at least 25%,preferably at least 50%, at least 60%, at least 75%, at least 85%, atleast 95%, or at least 99%. Further, antibodies, compositions, orcombination therapies administered according to the methods of theinvention can be tested for their ability reduce the hospitalizationperiod of humans suffering from fungal respiratory infection by at least60%, preferably at least 75%, at least 85%, at least 95%, or at least99%. Techniques known to those of skill in the art can be used toanalyze the function of the antibodies, compositions, or combinationtherapies of the invention in vivo.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of an antibody, acomposition, a combination therapy disclosed herein for prevention,treatment, management, and/or amelioration of disease or disorderassociated with or characterized by aberrant expression and/or activityof an IL-9 polypeptide, a disease or disorder associated with orcharacterized by aberrant expression and/or activity of the IL-9R or oneor more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof.

5.7.3. Toxicity Assays

The toxicity and/or efficacy of the prophylactic and/or therapeuticprotocols of the instant invention can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Therapies that exhibit large therapeutic indices are preferred. Whiletherapies that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such agents to the siteof affected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any therapy used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of an antibody, acomposition, a combination therapy disclosed herein for a disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof.

5.8. Diagnostic Uses of Antibody Formulations

Antibodies (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof) of the liquid formulationsof the invention that immunospecifically bind to an IL-9 polypeptide canbe used for diagnostic purposes to detect, diagnose, prognose, ormonitor a disease or disorder associated with or characterized byaberrant expression and/or activity of an IL-9 polypeptide, a disease ordisorder associated with or characterized by aberrant expression and/oractivity of the IL-9R or one or more subunits thereof, an autoimmunedisease, an inflammatory disease, a proliferative disease, or aninfection (preferably, a respiratory infection), or one or more symptomsthereof. The invention provides for the detection of aberrant expressionof IL-9 comprising: (a) assaying the expression of IL-9 in a biologicalsample from an individual using one or more antibodies of the liquidformulations of the invention that immunospecifically binds to an IL-9polypeptide; and (b) comparing the level of IL-9 with a standard levelof IL-9, e.g., in normal biological samples, whereby an increase ordecrease in the assayed level of IL-9 compared to the standard level ofIL-9 is indicative of a disease or disorder associated with orcharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disease or disorder associated with or characterized byaberrant expression and/or activity of the IL-9R or one or more subunitsthereof, an autoimmune disease, an inflammatory disease, a proliferativedisease, or an infection (preferably, a respiratory infection), or oneor more symptoms thereof. In specific embodiments, aberrant expressionlevel of IL-9 is indicative of an autoimmune disorder or a disease orcondition associated therewith. In another specific embodiment, anaberrant expression level of IL-9 is indicative of an inflammatorydisorder or a disease or condition associated therewith, such as asthma.In preferred embodiments, an aberrant expression level of IL-9 isindicative of a respiratory infection, such as, but not limited to RSV,PVI, or hMPV.

In preferred embodiments, the labeled antibodies of the liquidformulations of the invention that immunospecifically bind to IL-9 areused for diagnostic purposes to detect, diagnose, prognose, or monitor arespiratory infection, preferably RSV infection, PIV infection, or hMPV.The invention provides methods for the detection of a respiratoryinfection, comprising: (a) assaying the expression of IL-9 in cells or atissue sample of a subject using one or more antibodies thatimmunospecifically bind to IL-9; and (b) comparing the level of IL-9with a control level, e.g., levels in normal tissue samples notinfected, whereby an increase in the assayed level of IL-9 compared tothe control level of IL-9 is indicative of a respiratory infection.

Antibodies of the liquid formulations of the invention can be used toassay IL-9 levels in a biological sample using classicalimmunohistological methods as described herein or as known to those ofskill in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol.101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096).Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of IL-9 in an animal,preferably a mammal, and most preferably a human. In one embodiment,diagnosis comprises: a) administering (for example, parenterally,subcutaneously, or intraperitoneally) to a subject an effective amountof a labeled antibody (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof) of the liquidformulations of the invention that immunospecifically binds to an IL-9polypeptide; b) waiting for a time interval following the administeringfor permitting the labeled antibody to preferentially concentrate atsites in the subject where IL-9 is expressed (and for unbound labeledmolecule to be cleared to background level); c) determining backgroundlevel; and d) detecting the labeled antibody in the subject, such thatdetection of labeled antibody (including antibody fragment thereof)above the background level and above or below the level observed in aperson without the disease or disorder indicates that the subject has aparticular disease or disorder associated with aberrant expression ofIL-9. Background level can be determined by various methods including,comparing the amount of labeled molecule detected to a standard valuepreviously determined for a particular system. Aberrant expression ofIL-9 can occur particularly in lymphoid and myeloid cell types. A moredefinitive diagnosis of respiratory infection may allow healthprofessionals to employ preventive measures or aggressive treatmentearlier and thereby prevent the development or further progression ofthe infection.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99Tc. The labeled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds, MassonPublishing Inc. (1982). Depending on several variables, including thetype of label used and the mode of administration, the time intervalfollowing the administration for permitting the labeled molecule topreferentially concentrate at sites in the subject and for unboundlabeled molecule to be cleared to background level is 6 to 48 hours, 6to 24 hours, or 6 to 12 hours. In another embodiment the time intervalfollowing administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disorder, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled IL-9 antibody can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

In a specific embodiment, the IL-9 antibody is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the IL-9 antibody is labeled with a fluorescentcompound and is detected in the patient using a fluorescence responsivescanning instrument. In another embodiment, the IL-9 antibody is labeledwith a positron emitting metal and is detected in the patient usingpositron emission-tomography. In yet another embodiment, the IL-9antibody is labeled with a paramagnetic label and is detected in apatient using magnetic resonance imaging (MRI).

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) maybe utilized for immunophenotyping of cell lines and biological samplesby their IL-9 expression or IL-9 receptor expression. Various techniquescan be utilized using the antibodies, fragments, or variants of theliquid formulations of the invention to screen for cellular populations(that express IL-9 and/or IL-9 receptor, particularly immune cells,i.e., T and B lymphocytes, mast cells, eosinophils, macrophages,neutrophils and epithelial cells or IL-9 receptor, and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (see, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.,minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

5.9. Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with a liquid formulation of the invention. In aspecific embodiment, the liquid formulations of the invention compriseantibodies (including antibody fragments thereof) recombinantly fused orchemically conjugated to another moiety, including but not limited to, aheterologous protein, a heterologous polypeptide, a heterologouspeptide, a large molecule, a small molecule, a marker sequence, adiagnostic or detectable agent, a therapeutic moiety, a drug moiety, aradioactive metal ion, a second antibody, and a solid support. Theinvention also provides a pharmaceutical pack or kit comprising in oneor more first containers a liquid formulation of the invention and inone or more second containers one or more other prophylactic ortherapeutic agents useful for the prevention, management or treatment ofa disease or disorder associated with or characterized by aberrantexpression and/or activity of an IL-9 polypeptide, a disease or disorderassociated with or characterized by aberrant expression and/or activityof the IL-9R or one or more subunits thereof, an autoimmune disease, aninflammatory disease, a proliferative disease, or an infection(preferably, a respiratory infection), or one or more symptoms thereof.In a preferred embodiment, the liquid formulations of the invention areformulated in single dose vials as a sterile liquid containing 10 mMhistidine buffer at pH 6.0 and 150 mM sodium chloride. Each 1.0 mL ofsolution contains 100 mg of an IL-9 antibody (including antibodyfragment thereof), 1.6 mg of histidine and 8.9 mg of sodium chloride.During the manufacturing process, the pH of the formulation buffer isadjusted to 6.0 using hydrochloric acid. The formulations of theinvention may be supplied in 3 cc USP Type I borosilicate amber vials(West Pharmaceutical Serices—Part No. 6800-0675) with a target volume of1.2 mL. Optionally associated with such container(s) can be a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises a liquid formulation of theinvention, in one or more containers. In another embodiment, a kitcomprises a liquid formulation of the invention, in one or morecontainers, and one or more other prophylactic or therapeutic agentsuseful for the prevention, management or treatment of a disease ordisorder associated with or characterized by aberrant expression and/oractivity of an IL-9 polypeptide, a disease or disorder associated withor characterized by aberrant expression and/or activity of the IL-9R orone or more subunits thereof, an autoimmune disease, an inflammatorydisease, a proliferative disease, or an infection (preferably, arespiratory infection), or one or more symptoms thereof, in one or moreother containers. In a specific embodiment, the antibodies (includingantibody fragments thereof) included in said liquid formulations is 4D4,4D4H2-1 D11, 4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2,7F3com-3H5, or 7F3com-3D4 or an antigen-binding fragment. In analternative embodiment, the antibody (including antibody fragmentthereof) included in said liquid formulations is not 4D4, 4D4H2-1 D11,4D4com-XF-9, 4D4com-2F9, 7F3, 71A10, 7F3 22D3, 7F3com-2H2, 7F3com-3H5,or 7F3com-3D4 or an antigen-binding fragment thereof. Preferably, thekit further comprises instructions for preventing, treating, managing orameliorating a disorder (e.g., using the liquid formulations of theinvention alone or in combination with another prophylactic ortherapeutic agent), as well as side effects and dosage information formethod of administration.

5.10. Articles of Manufacture

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In thecase of dosage forms suitable for parenteral administration the activeingredient, e.g., an antibody of the invention that immunospecificallybinds to an IL-9 polypeptide, is sterile and suitable for administrationas a particulate free solution. In other words, the inventionencompasses both parenteral solutions and lyophilized powders, eachbeing sterile, and the latter being suitable for reconstitution prior toinjection. Alternatively, the unit dosage form may be a solid suitablefor oral, transdermal, intransal, or topical delivery.

In a preferred embodiment, the unit dosage form is suitable forintravenous, intramuscular, intranasal, oral, topical or subcutaneousdelivery. Thus, the invention encompasses solutions, preferably sterile,suitable for each delivery route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, total lymphocyte, mast cell counts, T cell counts, IgEproduction, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises a liquid formulation containing an antibody thatimmunospecifically binds to IL-9 and wherein said packaging materialincludes instruction means which indicate that said antibody can be usedto prevent, manage, treat, and/or ameliorate one or more symptomsassociated with a disorder associated with aberrant expression and/oractivity of an IL-9 polypeptide, a disorder associated with aberrantexpression and/or activity of an IL-9R or one or more subunits thereof,an autoimmune disorder, an inflammatory disorder, a proliferativedisorder, an infection (preferably, a respiratory infection), or one ormore symptoms thereof by administering specific doses and using specificdosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a liquid formulation containing an antibody thatimmunospecifically binds to an IL-9 polypeptide and the otherpharmaceutical agent comprises a second, different antibody thatimmunospecifically binds to an IL-9 polypeptide, and wherein saidpackaging material includes instruction means which indicate that saidagents can be used to treat, prevent and/or ameliorate a disorderassociated with aberrant expression and/or activity of an IL-9polypeptide, a disorder associated with aberrant expression and/oractivity of an IL-9R or one or more subunits thereof, an autoimmunedisorder, an inflammatory disorder, a proliferative disorder, aninfection (preferably, a respiratory infection), or one or more symptomsthereof by administering specific doses and using specific dosingregimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a liquid formulation containing an antibody thatimmunospecifically binds to an IL-9 polypeptide and the otherpharmaceutical agent comprises a prophylactic or therapeutic agent otherthan an antibody that immunospecifically binds to an IL-9 polypeptide,and wherein said packaging material includes instruction means whichindicate that said agents can be used to treat, prevent and/orameliorate one or more symptoms associated with a disorder associatedwith aberrant expression and/or activity of an IL-9 polypeptide, adisorder associated with aberrant expression and/or activity of an IL-9Ror one or more subunits thereof, an autoimmune disorder, an inflammatorydisorder, a proliferative disorder, an infection (preferably, arespiratory infection), or one or more symptoms thereof by administeringspecific doses and using specific dosing regimens as described herein.

The present invention provides that the adverse effects that may bereduced or avoided by the methods of the invention are indicated ininformational material enclosed in an article of manufacture for use inpreventing, treating and/or ameliorating one or more symptoms associatedwith an autoimmune disorder, an inflammatory disorder or an infection.Adverse effects that may be reduced or avoided by the methods of theinvention include, but are not limited to, vital sign abnormalities(fever, tachycardia, bardycardia, hypertension, hypotension),hematological events (anemia, lymphopenia, leukopenia,thrombocytopenia), headache, chills, dizziness, nausea, asthenia, backpain, chest pain (chest pressure), diarrhea, myalgia, pain, pruritus,psoriasis, rhinitis, sweating, injection site reaction, andvasodilatation. Since antibodies of the invention thatimmunospecifically bind to an IL-9 polypeptide may be immunosuppressive,prolonged immunosuppression may increase the risk of infection,including opportunistic infections. Prolonged and sustainedimmunosuppression may also result in an increased risk of developingcertain types of cancer.

Further, the information material enclosed in an article of manufacturefor use in preventing, treating, managing, and/or ameliorating disordercharacterized by aberrant expression and/or activity of an IL-9polypeptide, a disorder characterized by aberrant expression and/oractivity of an IL-9R or one or more subunits thereof, an inflammatorydisorder, an autoimmune disorder, a proliferative disorder, or aninfection (preferably, a respiratory infection) or one or more symptomsthereof can indicate that foreign proteins may also result in allergicreactions, including anaphylaxis, or cytosine release syndrome. Theinformation material should indicate that allergic reactions may exhibitonly as mild pruritic rashes or they may be severe such as erythroderma,Stevens-Johnson syndrome, vasculitis, or anaphylaxis. The informationmaterial should also indicate that anaphylactic reactions (anaphylaxis)are serious and occasionally fatal hypersensitivity reactions. Allergicreactions including anaphylaxis may occur when any foreign protein isinjected into the body. They may range from mild manifestations such asurticaria or rash to lethal systemic reactions. Anaphylactic reactionsoccur soon after exposure, usually within 10 minutes. Patients mayexperience paresthesia, hypotension, laryngeal edema, mental statuschanges, facial or pharyngeal angioedema, airway obstruction,bronchospasm, urticaria and pruritus, serum sickness, arthritis,allergic nephritis, glomerulonephritis, temporal arthritis, oreosinophilia.

6. EXAMPLES

6.1. Antibody Purification and Antibody Formulation

The following section describes a method for purifying antibodies to beused in the formulations of the invention (see FIG. 16).

6.1.1. Buffer Components and Equipment

Buffers, process solutions and cleaning solutions are prepared withwater for injection (WFI). Buffers are tested for bioburden andendotoxin.

Buffers and Process Solutions

-   -   0.1 M citric acid    -   10 mM sodium citrate, 80 mM NaCl, pH 4.6    -   25 mM sodium phosphate, pH 6.5    -   20 mM Tris-HCl, 40 mM NaCl, pH 7.5    -   0.5 M sodium phosphate, pH 6.5    -   5 mM sodium phosphate, 40 mM NaCl, pH 6.5    -   50 mM Glycine-HCl, 30 mM NaCl, pH 2.5    -   50 mM Glycine-HC, pH 2.35    -   1.0 M Tris base

Cleaning and Storage Solutions

-   -   Water for Injection (WFI)    -   1.0 N NaOH    -   0.1 N NaOH    -   20% (v/v) ethanol    -   0.5 N NaOH, 400 ppm sodium hypochlorite

Formulation Buffers

-   -   10 mM Histidine, 150 mM NaCl, pH 6.0    -   4 M sodium chloride

Equipment (Substitutions with Equivalent Performing Materials areAcceptable)

-   -   300 kg scale    -   Conductivity meter    -   Stir plate    -   pH meter    -   Vessels: Appropriately sized Stedim™ bags, buffer tanks, PETG        Bottles    -   Watson Marlow 1700 peristaltic pump    -   Wedgewood UV, pH, conductivity unit    -   Amersham Pharmacia chromatography controller    -   Packed POROS HS50 cation exchange gel    -   Packed Pharmacia rProtein A affinity gel    -   Packed POROS HQ anion exchange gel    -   Sterile, depyrogenated silicone tubing    -   Integritest Filter Integrity Tester II    -   Sterile Asahi Planova 20 N membrane viral removal filter    -   Millipore 0.2 micron Durapore filter    -   Millipore Multimedia filter    -   CUNO 60LP, 10/60 SP filter    -   CUNO filter housing    -   Class 100 hood

6.1.2. Purification and Formulation of the Antibodies

FIG. 16 outlines the process steps for the purification and formulationof the antibodies (including antibody fragments thereof) of theinvention. The purification process comprises three chromatographysteps, a nanofiltration step, a low pH treatment step, and formulation.These steps are designed to remove host cell proteins, DNA and cellculture components such as BSA and transferrin. In addition, the processincludes steps to control bioburden and endotoxin and to remove andinactivate viruses.

6.1.2.1. Conditioned Medium (Steps 1 to 6)

Conditioned medium from a single cell culture lot or pooled frommultiple cell culture lots is purified as a single lot. The combinationof multiple cell culture lots into one purification lot is performed inorder to utilize downstream processing steps sized for a single lot sizeand to decrease the number of purification lots. For example, becausethe working volumes of 130 L and 250 L cell culture bioreactors areapproximately 100 L and 200 L, respectively, these two cell culture lotscould be pooled and run as one 300 L purification lot. Process productsamples are analyzed for DNA using a PicoGreen or a quantitative PCRassay to detect DNA. Protein concentration is determined either by aProtein A bindable HPLC assay or by UV absorbance at 280 nm.Product-containing process streams are monitored for endotoxin andbioburden. Column effluents are monitored for endotoxin. A descriptionof each step is summarized below.

6.1.2.2. Conditioned Medium Adjustment and Filtration (Step 7)

The conditioned medium is adjusted to pH 4.6±0.2 with 0.1 M citric acid.The adjusted conditioned medium is then filtered using a CUNO filterin-line with a Millipore 0.2 micron Durapore filter.

6.1.2.3. Cation Exchange Chromatography Step (Step 8)

The adjusted and filtered conditioned medium is loaded onto a cationexchange column that has been equilibrated with 10 mM sodium phosphate,80 mM sodium chloride, pH 4.6. The bound antibody is washed using thesame buffer. The column is then washed with 25 mM sodium phosphate pH6.5 to remove process impurities, especially BSA. The product is elutedusing 20 mM Tris-HCl buffer, 40 mM NaCl, pH 7.5. Following elution ofthe product, the column is cleaned with 1.0 N NaOH and stored in 0.1 NNaOH at room temperature.

6.1.2.4. rProtein A Chromatograph (Step 9)

The cation exchange product is loaded directly onto a rProtein A columnequilibrated with 20 mM Tris-HCl buffer, 40 mM NaCl, pH 7.5. Followingloading, the column is washed with the equilibration buffer, and theproduct is eluted with 50 mM glycine, 30 mM NaCl, pH 3.2. The rProtein Aproduct is neutralized to pH 6.5±0.2 with 1.0 M Tris base. Thischromatography step removes additional process-related impurities. Atthe end of the step, the column is washed with equilibration buffer,cleaned with 0.1 N NaOH, washed with equilibration buffer and stored in20% (v/v) ethanol at room temperature.

6.1.2.5. Anion Exchange Chromatography (Step 10)

This chromatographic step is the final step designed to remove any tracelevels of process-related impurities. The column is equilibrated with0.5 M sodium phosphate, pH 6.5 followed by equilibration with 5 mMsodium phosphate, 40 mM sodium chloride, pH 6.5. Under these conditions,the neutralized rProtein A product is loaded onto the equilibrated anionexchange column, and under these conditions, the product is recovered inthe non-bound fraction and the process-related impurities are retainedin the column. The column is cleaned with 1.0 N NaOH and stored in 0.1 NNaOH at room temperature.

6.1.2.6. Nanofiltration (Step 11)

The anion exchange product is filtered through a sterile Planova™ 20 Nmembrane (pore size=20 nm) that is prepared by flushing first with WFIand then with 5 mM sodium phosphate, 40 mM sodium chloride pH 6.5. Afterthe product is filtered, the filter is chased with a small volume of 5mM sodium phosphate, 40 mM sodium chloride, pH 6.5 to maximize productrecovery. After filtration the nanofilter is integrity tested.

6.1.2.7. Low pH Treatment (Step 12)

The pH of the nanofiltered product is adjusted to 3.4±0.1 with 50 mMglycine, pH 2.35 and held at this pH for 30±10 minutes. After low pHtreatment, the product pH is adjusted to 6.5±0.2 with 1.0 M Tris base.

6.1.2.8. Formulation of Anti-IL-9 Antibodies

Sodium chloride (150 mM) is added to the antibody purified as in steps1-12 above using a 4 M NaCl stock solution. It is then 0.2 micronfiltered and concentrated to 20 g/L using tangential flow filtrationwith a 30 kDa membrane. The product is then diafiltered into theformulation buffer (10 mM Histidine, 150 mM NaCl, pH 6.0), using aminimum of five buffer exchanges. The product is then concentrated to100 g/L and filtered through a 1.2/0.2 micron membrane into sterileStedim® storage bags.

6.2. Monitoring Antibody Stability and Aggregation of AntibodyFormulations

6.2.1. Reference Standard Stability

The stability of the 7F3com-2H2 Reference Standard (purified as inSection 6.1 above) was tested. Following low pH treatment, the antibodycomposition was adjusted to pH 6.5, concentrated to 11 mg/ml bytangential flow filtration, buffer exchanged into 10 mM Histidine pH 6.0buffer and filtered through a 0.22 micron filter yielding an anti-IL-9antibody Reference Standard at a protein concentration of 11.3 mg/ml.

The following stability-indicating tests were used to assess thestability of the anti-IL-9 antibody Reference Standard: appearance,native isoelectric focusing (IEF), reducing and non-reducing gelelectrophoresis, HPSEC, IL-9 binding ELISA, and protein concentration byA₂₈₀. These assays represent the primary identity, purity, and potencytests and are capable of detecting aggregates, proteolytic degradation,and loss of potency.

Tests were done at −70° C., 2-8° C., 38-42° C., or after threefreeze-thaw cycles. The −70° C. stability condition was chosen becausereference standards are typically stored at this temperature. The threefreeze-thaw cycle study was done to determine the robustness of theReference Standard material stored at −70° C. in the event ofinadvertent thawing and to assess if there is any adverse effect withrepeated freeze/thaw cycles. The 2-8° C. condition was chosen toevaluate possible alternative storage conditions. The 38-42° C.accelerated temperature condition was chosen to demonstrate that thechosen assays are stability-indicating and to monitor the stability ofthe molecule under stress conditions.

Table 5, infra, shows the stability testing results for the 7F3com-2H2Reference Standard. All test results for the 2-8° C. and the 3×freeze-thaw conditions listed in the table were within specifications.Unsatisfactory percent purity results were obtained for the two andthree-month time points of condition D. After 3-month storage at 38-42°C., the percent purity by HPSEC dropped to 93.6%. The non-reduced gelelectrophoresis profile at the same condition at the two and three-monthtime points was not consistent when compared to the initial ReferenceStandard profile. The isoelectric focusing (IEF) profile for the two andthree-month time point at 38-42° C. was not consistent when compared tothe initial Reference Standard (stored at −70° C.) due to the presenceof a new acidic band that is not seen in the Reference Standard.

TABLE 5 Stability Results for the 7F3com-2H2 Reference Standard

6.2.2. Antibody Formulation Stability

The stability of the 7F3com-2H2 antibody formulation (described inSection 6.1) is also monitored by the same stability-indicating assaysused to evaluate the stability of the 7F3com-2H2 Reference Standard.These assays are performed at intervals indicated in Table 6, infra.

TABLE 6 Stability Testing Plan for the 7F3com-2H2 Antibody FormulationTime (months) Condition 0 0.25 0.50 0.75 1 1.5 2 3 6 9 12 18 24 36 B(2-8° C.) X X X X X X X X X X X X X X X = assays are performed at thistime point;

The 2-8° C. condition was chosen because the 7F3com-2H2 antibodycomposition in the purification process is adjusted to pH 6.5, andstored at this temperature prior to formulation, concentration, andfill. The storage time is not anticipated to exceed 12 months; however,the stability is continually monitored for at least 36 months in theevent that prolonged storage at 2-8° C. is needed.

Table 7, infra, shows the stability testing results obtained for the7F3com-2H2 antibody formulation. The 2-8° C. storage condition samplesmet specifications through the three-month time point.

TABLE 7 Stability Results for the 7F3com-2H2 Antibody Formulation TotalIEF Gel electrophoresis Time Protein (non- (non- IL-9 Binding Conditionpoint (A280) reduced) (reduced) reduced) HPSEC Appearance ELISADescription (month) (mg/ml) (Pass/Fail) (Pass/Fail) (% Purity)(Pass/Fail) (% Purity) (% Aggregate) (Pass/Fail) Parallelism (ED50)Condition B: 0 2.3 Pass Pass 98.5 Pass 99.5 0.5 Pass Pass 15.5 2-8° C.Condition B: 0.25 2.4 Pass Pass 97.7 Pass 99.5 0.5 Pass Pass 10.8 2-8°C. Condition B: 0.50 2.6 Pass Pass 98.2 Pass 99.2 0.5 Pass Pass 28.02-8° C. Condition B: 0.75 2.3 Pass Pass 98.1 Pass 99.5 0.5 Pass Pass26.4 2-8° C. Condition B: 1 2.3 Pass Pass 99.7 Pass 99.5 0.5 Pass Pass10.2 2-8° C. Condition B: 1.5 2.4 Pass Pass 98.0 Pass 99.5 0.5 Pass Pass16.7 2-8° C. Condition B: 2 2.4 Pass Pass 98.5 Pass 99.4 0.6 Pass Pass20.3 2-8° C. Condition B: 3 2.4 Pass Pass 98.2 Pass 99.5 0.5 Pass Pass11.0 2-8° C.

6.3. Compositions and Formulations of Antibodies

6.3.1. Description and Composition

F3com-2H2 antibodies are formulated in single dose vials as a sterileliquid containing 10 mM histidine buffer at pH 6.0 and 150 mM sodiumchloride. Each 1.0 mL of solution contains 100 mg of protein, 1.6 mg ofhistidine and 8.9 mg of sodium chloride in water for injection. Duringthe manufacturing process, the pH of the formulation buffer is adjustedto pH 6.0 using hydrochloric acid.

6.3.2. Formulation Development

Formulation studies were designed to evaluate chemical and physicalproperties that influence protein stability and solubility. The goal wasto determine the most suitable conditions for long-term storage of theproduct. Studies were divided into three main areas: preformulation,concentration and stability. Preformulation biophysical studies, such asdifferential scanning calorimetry, were used to evaluate the effects oftemperature and pH on the secondary and tertiary structure of theprotein. Concentration studies were undertaken to provide information onnative protein-protein interactions during a concentration step, and theimpact of high concentration on long-term storage. Solution pH studieswere designed to examine buffers over a physiologically useful pH rangein order to achieve optimal solubility of the protein at highconcentration. Based on data generated, a formulation of 7F3com-2H2 at100 mg/mL concentration in 10 mM histidine, pH 6.0 that contains 150 mMNaCl was developed as providing optimal stability and solubility.

6.3.2.1. Container Closure System

The antibody formulations are supplied at 100 mg/ml in 3 cc USP Type Iborosilicate amber vials. The target fill volume is 1.2 mL, whichrepresents a 20% overage and meets USP guidelines. A description of eachcomponent of the container closure system is listed below.

Vials

-   -   West Pharmaceutical Services—Part # 6800-0675 (Manufacturer:        Schott) 3 cc, 13 mm amber serum/lyo, USP/EP Type I Borosilicate,        blowback (Purform)

Stoppers

-   -   West Pharmaceutical Services—Part # 1012-4635, 13 mm 4432/50        gray chlorobutyl, Teflon-coated, Westar RS Silicone level 3

Overseals

-   -   West Pharmaceutical Services, 13 mm Flip-Off TruEdge

6.4. Stability Testing of Vialed Antibody Formulations

The 7F3com-2H2 liquid formulation was vialed at a concentration of 100mg/mL in 10 mM Histidine-HCl, 150 mM sodium chloride, pH 6.0 buffer andtested for stability. Vials were stored at 2-8° C. (condition B), 20-24°C. (condition C) and 38-42° C. (condition D). For this study,appearance, protein concentration by A₂₈₀, reducing and non-reducing gelelectrophoresis, native IEF, IL-9 binding ELISA, and HPSEC wereevaluated on the upright vial condition. Time points for each conditionare listed in Table 8, infra.

TABLE 8 Stability Testing Plan for Vialed 7F3com-2H2 Formulation TimeCondi- (Months tion 0 0.5 0.75 1 1.5 2 3 6 9 12 18 24 36 B X X X X X X XX X X X X X (2-8° C.) C X X X X X X X X NP NP NP NP NP (20-24° C.) D X XX X X X X X NP NP NP NP NP (38-42° C.) X = testing preformed at thistime point; NP—test not performed at this time point. * = upright vials.

Test results for the initial time point through the three-month timepoints for all three conditions are listed Table 9, infra. The vialedproduct was stable for at least three months when stored at 2-8° C. and20-24° C. storage temperatures. No discernible changes in stabilityassays were seen after two weeks of storage at the 38-42° C. condition.After three weeks of storage at the elevated temperature of 38-42° C.,the percent purity determined by the HPSEC method dropped to 94.9%.Unsatisfactory percent purity results were obtained for the three weeks,one-month, one and a half-month, two-month, and three-month time pointsof condition D. The chromatographic profile showed both aggregate andlower molecular weight degradation products. In addition, thenon-reducing gel electrophoresis profile for the one-month, one and ahalf-month, two-month, and three-month time points of condition D wasnot consistent when compared to the Reference Standard due to thepresence of small peaks that are not seen in the Reference Standardprofile. The IEF profile for the one and half-month, two-month, andthree-month time points at 38-42° C. was not consistent when compared tothe Reference Standard due to the presence of a new acidic band that isnot seen in the Reference Standard. These data indicate there is asufficient stability of 7F3com-2H2 at the recommended storagetemperature.

TABLE 9 Stability Results for Vialed 7F3com-2H2 Antibody Formulation

A second lot of the 7F3com-2H2 antibody formulation was vialed at aconcentration of 100 mg/mL in 10 mM Histidine-HCl, 150 mM sodiumchloride, pH 6.0 buffer and placed on a stability protocol. Thestability plan for 7F3com-2H2 antibody formulation is outlined in Table10, infra. The stability-indicating assays chosen are reducing andnon-reducing gel electrophoresis, native IEF, IL-9 binding ELISA, andHPSEC. The general assays of A280 and appearance are also be performed.Container/closure integrity testing are conducted at 0, 6, 12, 24, and36-month time points for condition B, and at 0, and 6-month time pointsfor condition C. This test may also be performed on vials for conditionD at 0 and 3-month time points. All tests for condition B are performedonly on the inverted vial condition. The upright vial orientation areused for the accelerated temperature conditions (C and D).

TABLE 10 Stability Testing Plan for Vialed Antibody Formulations Time(Months Condition 0 1 2 3 6 9 12 18 24 36 B inverted vial, (2-8° C.) XNP NP X X X X X X X B upright vial, (2-8° C.) NP NP NP NP NP NP NP NP NPNP C inverted vial X X X X X NP NP NP NP NP (20-24° C.) D upright vial XX X X NP NP NP NP NP NP (38-42° C.) *The upright vial orientation isanalyzed in the event of a stability failure at condition B (2-8° C.)for the inverted vial orientation. Container/closure integrity testingis performed on inverted vials from condition B & C at the 0, 6, 12, 24,and 36-month time points only, and at the 0 and 3-month time points forcondition D. X = testing performed at this time point; NP = test notperformed at this time point.

7. EOUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. A sterile antibody formulation comprising an aqueous carrier, between1 mM and 100 mM of histidine, and 50 mg/ml or higher of an antibody orantibody fragment that immunospecifically binds to an interleukin-9polypeptide, wherein the antibody or antibody fragment comprises a VHdomain comprising the amino acid sequence of SEQ ID NO:27 and a VLdomain comprising the amino acid sequence of SEQ ID NO:28.
 2. Apharmaceutical unit dosage form suitable for parenteral administrationto a human which comprises an antibody formulation of claim 1 in asuitable container.
 3. The pharmaceutical unit dosage form of claim 2,wherein the antibody formulation is administered intravenously,subcutaneously, or intramuscularly.
 4. A pharmaceutical unit dosage formsuitable for aerosol administration to a human which comprises anantibody formulation of claim 1 in a suitable container.
 5. Thepharmaceutical unit dosage of claim 4, wherein the antibody formulationis suitable for intranasal administration.
 6. An antibody formulationwhich is produced by lyophilizing the aqueous antibody formulation ofclaim
 1. 7. A hermetically sealed container containing the formulationof claim
 1. 8. A hermetically sealed container containing theformulation of claim
 6. 9. The hermetically sealed container of claim 8having sufficient volume for reconstitution with a pharmaceuticallyacceptable carrier.
 10. The hermetically sealed container of claim 9,wherein said carrier is water for injection, 5% dextrose in water orsaline.
 11. The hermetically sealed container of claim 10, wherein saidcontainer maintains a sterile environment and allows reconstitution ofthe formulation without loss of sterility.
 12. A kit comprising in oneor more containers the antibody formulation of claim 1, and instructionsfor use of the formulation, wherein said formulation is substantiallyfree of surfactant.
 13. The kit of claim 12, wherein the aqueous carrieris distilled water.
 14. The kit of claim 12, wherein the formulation isproduced by lyophilizing the aqueous antibody formulation.
 15. The kitof claim 12, wherein the antibody or antibody fragment is at aconcentration of 95 mg/ml.
 16. The formulation of claim 1, comprisingNaCl.
 17. The formulation of claim 16, wherein the NaCl is at aconcentration in the range from about 100 mM to about 200 mM.
 18. Theformulation of claim 17, wherein the NaCl is at a concentration of 150mM.
 19. The formulation of claim 1, wherein said formulation issubstantially free of surfactants, sugars, sugar alcohols, and aminoacids other than histidine.
 20. The formulation of claim 19, whereinsaid amino acids other than histidine have a pKa value of less than 5 orabove
 7. 21. The formulation of claim 1, wherein the aqueous carrier isdistilled water.
 22. The formulation of claim 1 further comprisingglycine at a concentration in the range from about 1 to about 10 mM. 23.The formulation of claim 1, wherein the histidine is at a concentrationin the range from about 5 to about 25 mM.
 24. The formulation of claim23, wherein the histidine is at a concentration of 10 mM.
 25. Theformulation of claim 1, wherein the formulation has a pH in the rangebetween 5.0 and 7.0.
 26. The formulation of 25, wherein said formulationhas a pH of 6.0.
 27. The formulation of claim 1 further comprising anexcipient.
 28. The formulation of claim 27, wherein the excipient is asaccharide.
 29. The formulation of claim 27, wherein the excipient is apolyol.
 30. The formulation of claim 1, wherein the antibody or antibodyfragment is at a concentration of at least 95 mg/ml.
 31. The formulationof claim 30, wherein the antibody or antibody fragment is at aconcentration of at least 100 mg/ml.
 32. The formulation of claim 31,wherein the antibody or antibody fragment is at a concentration of atleast 150 mg/ml.
 33. The formulation of claim 1, wherein the antibody orantibody fragment that immunospecifically binds to an interleukin-9polypeptide is stable during storage at 40° C. for at least 15 days asdetermined by high performance size exclusion chromatography (HPSEC).34. The formulation of claim 1, wherein the antibody or antibodyfragment that immunospecifically binds to an interleukin-9 polypeptideis stable during storage at about ambient temperature for at least 6months as determined by HPSEC.
 35. The formulation of claim 1, whereinthe antibody or antibody fragment that immunospecifically binds to aninterleukin-9 polypeptide is stable during storage at 4° C. for at least1.5 years as determined by HPSEC.
 36. The formulation of claim 33,wherein less than 5% of the antibody or antibody fragment forms anaggregate during the storage as measured by HPSEC.
 37. The formulationof claim 34, wherein less than 5% of the antibody or antibody fragmentforms an aggregate during the storage as measured by HPSEC.
 38. Theformulation of claim 35, wherein less than 5% of the antibody orantibody fragment forms an aggregate during the storage as measured byHPSEC.
 39. The formulation of claim 36, wherein less than 2% of theantibody or antibody fragment forms an aggregate during the storage asmeasured by HPSEC.
 40. The formulation of claim 37, wherein less than 2%of the antibody or antibody fragment forms an aggregate during thestorage as measured by HPSEC.
 41. The formulation of claim 38, whereinless than 2% of the antibody or antibody fragment forms an aggregateduring the storage as measured by HPSEC.
 42. The formulation of claim39, wherein less than 1% of the antibody or antibody fragment forms anaggregate during the storage as measured by HPSEC.
 43. The formulationof claim 40, wherein less than 1% of the antibody or antibody fragmentforms an aggregate during the storage as measured by HPSEC.
 44. Theformulation of claim 41, wherein less than 1% of the antibody orantibody fragment forms an aggregate during the storage as measured byHPSEC.
 45. The formulation of claim 33, wherein the antibody or antibodyfragment retains at least 80% of binding ability to an interleukin-9polypeptide compared to a reference antibody representing the antibodyprior to the storage.
 46. The formulation of claim 34, wherein theantibody or antibody fragment retains at least 80% of binding ability toan interleukin-9 polypeptide compared to a reference antibodyrepresenting the antibody prior to the storage.
 47. The formulation ofclaim 35, wherein the antibody or antibody fragment retains at least 80%of binding ability to an interleukin-9 polypeptide compared to areference antibody representing the antibody prior to the storage. 48.The formulation of claim 45, wherein the antibody or antibody fragmentretains at least 85% of binding ability to an interleukin-9 polypeptidecompared to the reference antibody.
 49. The formulation of claim 46,wherein the antibody or antibody fragment retains at least 85% ofbinding ability to an interleukin-9 polypeptide compared to thereference antibody.
 50. The formulation of claim 47, wherein theantibody or antibody fragment retains at least 85% of binding ability toan interleukin-9 polypeptide compared to the reference antibody.
 51. Theformulation of claim 48, wherein the antibody or antibody fragmentretains at least 90% of binding ability to an interleukin-9 polypeptidecompared to the reference antibody.
 52. The formulation of claim 49,wherein the antibody or antibody fragment retains at least 90% ofbinding ability to an interleukin-9 polypeptide compared to thereference antibody.
 53. The formulation of claim 50, wherein theantibody or antibody fragment retains at least 90% of binding ability toan interleukin-9 polypeptide compared to the reference antibody.
 54. Theformulation of claim 51, wherein the antibody or antibody fragmentretains at least 95% of binding ability to an interleukin-9 polypeptidecompared to the reference antibody.
 55. The formulation of claim 52,wherein the antibody or antibody fragment retains at least 95% ofbinding ability to an interleukin-9 polypeptide compared to thereference antibody.
 56. The formulation of claim 53, wherein theantibody or antibody fragment retains at least 95% of binding ability toan interleukin-9 polypeptide compared to the reference antibody.
 57. Theformulation of claim 30, wherein the histidine is at a concentration of10 mM and wherein the aqueous carrier is distilled water, and whereinthe formulation further comprises NaCl at a concentration of 150 mM. 58.The formulation of claim 57, wherein the antibody or antibody fragmentthat immunospecifically binds to an interleukin-9 polypeptide is stableduring storage at 40° C. for at least 15 days as determined by highperformance size exclusion chromatography (HPSEC).
 59. The formulationof claim 57, wherein the antibody or antibody fragment thatimmunospecifically binds to an interleukin-9 polypeptide is stableduring storage at 4° C. for at least 1.5 years as determined by HPSEC.60. The formulation of claim 57, wherein less than 5% of the antibody orantibody fragment forms an aggregate during the storage as measured byHPSEC.
 61. The formulation of claim 57, wherein the antibody or antibodyfragment retains at least 80% of binding ability to an interleukin-9polypeptide compared to a reference antibody representing the antibodyprior to the storage.
 62. A sterile antibody formulation comprising anaqueous carrier, between 1 mM and 100 mM of histidine, and 50 mg/ml orhigher of an antibody or antibody fragment that immunospecifically bindsto an interleukin-9 polypeptide, wherein the antibody or antibodyfragment comprises: a) a VH CDR1 comprising the amino acid sequence ofSEQ ID NO:26, b) a VH CDR2 comprising the amino acid sequence of SEQ IDNO:61, c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3,d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:62, e) a VLCDR2 comprising the amino acid sequence of SEQ ID NO:63, and f) a VLCDR3 comprising the amino acid sequence of SEQ ID NO:20.
 63. Theformulation of claim 62, further comprising NaCl.
 64. The formulation ofclaim 63, wherein the NaCl is at a concentration of about 100 mM toabout 200 mM.
 65. The formulation of claim 64, wherein the NaCl is at aconcentration of 150 mM.
 66. The formulation of claim 62 furthercomprising glycine at a concentration of about 1 to about 10 mM.
 67. Theformulation of claim 62, wherein the histidine is at concentration ofabout 5 to about 25 mM.
 68. The formulation of claim 67, wherein thehistidine is at a concentration of 10 mM.
 69. The formulation of claim62, wherein said formulation is substantially free of surfactants,sugars, sugar alcohols, and amino acids other than histidine.
 70. Theformulation of claim 69, wherein said amino acids other than histidinehave a pKa value of less than 5 or above
 7. 71. The formulation of claim62, wherein the aqueous carrier is distilled water.
 72. The formulationof claim 62, wherein the formulation has a pH in the range between 5.0and 7.0.
 73. The formulation of claim 72, wherein said formulation has apH of 6.0.
 74. The formulation of claim 62, wherein the antibody orantibody fragment is at a concentration of at least 95 mg/ml.
 75. Theformulation of claim 74, wherein the antibody or antibody fragment is ata concentration of at least 100 mg/ml.
 76. The formulation of claim 75,wherein the antibody or antibody fragment is at a concentration of atleast 150 mg/mi.
 77. The formulation of claim 74, wherein the histidineis at a concentration of 10 mM and wherein the aqueous carrier isdistilled water, and wherein the formulation further comprises NaCl at aconcentration of 150 mM.
 78. The formulation of claim 77, wherein theantibody or antibody fragment that immunospecifically binds to aninterleukin-9 polypeptide is stable during storage at 40° C. for atleast 15 days as determined by high performance size exclusionchromatography (HPSEC).
 79. The formulation of claim 77, wherein theantibody or antibody fragment that immunospecifically binds to aninterleukin-9 polypeptide is stable during storage at 4° C. for at least1.5 years as determined by HPSEC.
 80. The formulation of claim 77,wherein less than 5% of the antibody or antibody fragment forms anaggregate during the storage as measured by HPSEC.
 81. The formulationof claim 77, wherein the antibody or antibody fragment retains at least80% of binding ability to an interleukin-9 polypeptide compared to areference antibody representing the antibody prior to the storage.